Amplification reporter with base-pairing oligomers

ABSTRACT

System, including methods, apparatus, and compositions, for performing amplification assays with an amplification reporter including a first oligomer and a second oligomer capable of base-pairing with one another below a melting temperature of the reporter. The reporter may have a detectable photoluminescence that is affected, such as reduced, by base-pairing of the first and second oligomers with one another. A target, such as a nucleic acid target sequence, may be amplified in at least one volume, such as a plurality of partitions, above the melting temperature, and photoluminescence of the reporter may be detected from the at least one volume below the melting temperature. A property of the target, such as a concentration of the target, may be determined based on the photoluminescence detected.

CROSS-REFERENCES TO PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/411,482, filed Jan. 20, 2017, now U.S. Pat. No. 10,604,789, which, inturn, is a continuation of U.S. patent application Ser. No. 14/457,863,filed Aug. 12, 2014, now U.S. Pat. No. 9,556,475, which, in turn, isbased upon and claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application Ser. No. 61/864,788, filed Aug. 12, 2013.Each of these priority patent applications is incorporated herein byreference in its entirety for all purposes.

CROSS-REFERENCES TO OTHER MATERIALS

This application incorporates by reference in their entireties for allpurposes the following materials: U.S. Pat. No. 7,041,481, issued May 9,2006; U.S. Patent Application Publication No. 2010/0173394 A1, publishedJul. 8, 2010; U.S. Patent Application Publication No. 2011/0217712 A1,published Sep. 8, 2011; U.S. Patent Application Publication No.2012/0152369 A1, published Jun. 21, 2012; U.S. Patent ApplicationPublication No. 2012/0194805 A1, published Aug. 2, 2012; U.S. patentapplication Ser. No. 14/171,754, filed Feb. 3, 2014; and Joseph R.Lakowicz, PRINCIPLES OF FLUORESCENCE SPECTROSCOPY (2^(nd) Ed. 1999).

INTRODUCTION

Digital assays generally rely on the ability to detect the presence oractivity of individual copies of an analyte in a sample. In an exemplarydigital assay, a sample is separated into a set of partitions, generallyof equal volume, with each containing, on average, less than about onecopy of the analyte. If the copies of the analyte are distributedrandomly among the partitions, some partitions should contain no copies,others only one copy, and, if the number of partitions is large enough,still others should contain two copies, three copies, and even highernumbers of copies. The probability of finding exactly 0, 1, 2, 3, ormore copies in a partition, based on a given average concentration ofanalyte in the partitions, may be described by a Poisson distribution.Conversely, the concentration of analyte in the partitions (and thus inthe sample) may be estimated from the probability of finding a givennumber of copies in a partition.

Estimates of the probability of finding no copies and of finding one ormore copies may be measured in the digital assay. Each partition can betested to determine whether the partition is a positive partition thatcontains at least one copy of the analyte, or is a negative partitionthat contains no copies of the analyte. The probability of finding nocopies in a partition can be approximated by the fraction of partitionstested that are negative (the “negative fraction”), and the probabilityof finding at least one copy by the fraction of partitions tested thatare positive (the “positive fraction”). The positive fraction or,equivalently, the negative fraction then may be utilized to determinethe concentration of the analyte in the partitions by Poissonstatistics.

Digital assays frequently rely on amplification of a nucleic acid targetin partitions to enable detection of a single copy of an analyte.Amplification of the target may be conducted via the polymerase chainreaction (PCR), to achieve a digital PCR assay. Amplification of thetarget can be detected optically from a probe included in the reaction.The probe may include an oligonucleotide conjugated to a fluorophore anda quencher. The oligonucleotide may be cut as the target is amplified,to separate the fluorophore from the quencher. As a result, lightemission from the fluorophore may be quenched more efficiently by thequencher in the absence of target amplification (a negative partition)than in the presence of target amplification (a positive partition).However, the difference in fluorescence between negative partitions andpositive partitions can vary with the assay configuration, oftendiminishing the ability to reliably distinguish the two types ofpartitions.

A simple and cost-effective approach is needed to minimize noise inprobe-based amplification assays, such as in digital PCR assays.

SUMMARY

The present disclosure provides a system, including methods, apparatus,and compositions, for performing amplification assays with anamplification reporter including a first oligomer and a second oligomercapable of base-pairing with one another below a melting temperature ofthe reporter. The reporter may have a detectable photoluminescence thatis affected, such as reduced, by base-pairing of the first and secondoligomers with one another. A target, such as a nucleic acid targetsequence, may be amplified in at least one volume, such as a pluralityof partitions, above the melting temperature, and photoluminescence ofthe reporter may be detected from the at least one volume below themelting temperature. A property of the target, such as a concentrationof the target, may be determined based on the photoluminescencedetected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary reporter composed ofbase-pairing oligomers (a “probe” and a “sink”) that collectivelyprovide a photoluminophore and an energy transfer partner, such as aquencher, for the photoluminophore, in accordance with aspects of thepresent disclosure.

FIG. 2 is a schematic comparing the levels of fluorescence produced bythe reporter of FIG. 1 while in a base-paired form, the probe of thereporter of FIG. 1 separated from the sink, and a degraded (cleaved)form of the probe resulting from target amplification, in accordancewith aspects of the present disclosure.

FIG. 3 is a schematic flowchart illustrating selected steps andconfigurations of an exemplary assay performed with the reporter of FIG.1, in accordance with aspects of the present disclosure.

FIG. 4 is a schematic flowchart of an exemplary assay performed with areporter composed of base-pairing oligomers, in accordance with aspectsof the present disclosure.

FIG. 5 is a schematic diagram of an exemplary system for performing theassay of FIG. 4, in accordance with aspects of the present disclosure.

FIG. 6 is a schematic comparing levels of fluorescence from (a) anexemplary reporter including a sink base-paired with a probe, with theprobe configured as a photoluminophore-labeled primer, and (b) the probeincorporated by primer extension into an amplicon that excludes the sinkfrom base-pairing interaction with the probe, in accordance with aspectsof the present disclosure.

FIG. 7 is a schematic flowchart illustrating selected steps andconfigurations of an exemplary assay performed with the reporter of FIG.6, in accordance with aspects of the present disclosure.

FIG. 7A is a graph showing an exemplary thermal profile for stages ofthe assay of FIGS. 6 and 7, and an exemplary status of the reporterduring at least a portion of each stage, in accordance with aspects ofthe present disclosure.

FIG. 8 is a schematic comparing levels of fluorescence produced by (a)yet another exemplary reporter including a probe and a sink thatbase-pair with one another, and (b) the probe of the reporter degradedas a result of target amplification, in accordance with aspects of thepresent disclosure.

FIG. 9 is a schematic comparing levels of fluorescence from (a) a highermelting-temperature version of the reporter of FIG. 6, and (b) apolymerase-extended version of a probe from the reporter, with the probeforming part of an amplicon and with a sink of the reporter cleaved as aresult of target amplification, in accordance with aspects of thepresent disclosure.

FIG. 9A is a schematic view of an exemplary reporter including a sinkhaving a quencher and base-paired with a probe, with the sink configuredto function as a polymerase-extendable primer for amplification, inaccordance with aspects of the present disclosure.

FIG. 10 is a schematic view of an exemplary reporter including a sinkbase-paired with a probe, with the probe arranged to quenchphotoluminescence intramolecularly as a result of binding to the sink,in accordance with aspects of the present disclosure.

FIG. 11 is a schematic view of an exemplary reporter including a sinkbound to a probe, with the probe arranged to quench photoluminescenceintermolecularly as a result of binding to the sink, in accordance withaspects of the present disclosure.

FIG. 12 is a schematic view of an exemplary reporter including a sinkbound to a probe, with a photoluminophore of the probe and a quencher ofthe sink each positioned intermediate the opposing ends of the probe orsink, in accordance with aspects of the present disclosure.

FIG. 13 is a schematic flowchart illustrating a strategy for assay of atarget region using at least two distinct probes, with at least one ofthe probes being a primer bound by a sink at room temperature andanother of the probes being self-quenched intramolecularly, inaccordance with aspects of the present disclosure.

FIG. 14 is another schematic flowchart illustrating another strategy forassay of a target region using at least two distinct probes, with eachof the probes being a primer bound by a different sink at roomtemperature, in accordance with aspects of the present disclosure.

FIG. 14A is still another schematic flowchart illustrating still anotherstrategy for assay of a target region using at least two distinctprobes, with each of the probes being a forward or reverse primer boundby a different sink at room temperature, in accordance with aspects ofthe present disclosure.

FIG. 15 is a schematic flowchart illustrating a strategy for selectivelyquenching emission of light from a primer dimer relative to a desiredamplicon in an amplification assay performed with aphotoluminophore-labeled primer as a probe, in accordance with aspectsof the present disclosure.

FIG. 16 is a plot of fluorescence amplitude measured for a series ofassays performed in droplets, and showing changes in the basalfluorescence of negative droplets, and differences in the fluorescenceamplitude between negative and positive droplets, for various targetsand with different levels of assay multiplexing.

FIG. 17 is a plot of fluorescence amplitude measured for a series ofassays performed in droplets, and showing changes in the basalfluorescence of negative droplets for various assay combinations,different levels of assay multiplexing, and/or with a different primerpair omitted from the assay.

FIG. 18 is a schematic view of a probe in an ideal singleplex assayversus a probe in a non-ideal singleplex or multiplex assay,illustrating a possible change in average spacing between aphotoluminophore and a quencher of the probe in the two assays, inaccordance with aspects of the present disclosure.

FIG. 19 is a plot of fluorescence amplitude measured for a series ofassays performed in droplets with five probes or one probe,respectively, in the absence and presence of a sink designed to positioncopies of one of the probes adjacent one another such that aphotoluminophore of one probe copy is quenched by a quencher of anotherprobe copy, in accordance with aspects of the present disclosure.

FIG. 20 is a schematic flowchart illustrating a pair of distinctstrategies for assay of overlapping target sequences in partitions(e.g., droplets), with the strategy on the left utilizing a probe actingas a primer and bound by a sink at room temperature, and the strategy onthe right utilizing a self-quenched probe positioned intermediate a pairof primers, in accordance with aspects of the present disclosure.

FIG. 21 is a plot of fluorescence data collected from droplets andcomparing the two strategies of FIG. 19 at different annealingtemperatures for thermocycling the droplets, in accordance with aspectsof the present disclosure.

FIG. 22 is a graph of target concentration determined from the data ofFIG. 21 for each of the sets of droplets of each assay strategy, inaccordance with aspects of the present disclosure.

FIG. 23 is a schematic of a strategy for performing a multiplex assay ofwild-type and mutant alleles of a K-Ras target in droplets, with probes,amplification primers, and a sink aligned with their prospective bindingsites in wild-type (WT) and mutant (MUT) templates, in accordance withaspects of the present disclosure.

FIG. 24 is a series of scatter plots of fluorescence data collected froma multiplex assay of a K-Ras target performed in droplets according tothe strategy of FIG. 23, with different amounts of the sink present inthe droplets, in accordance with aspects of the present disclosure.

FIG. 25 is a schematic of another strategy for performing a multiplexassay of wild-type and mutant alleles of a K-Ras gene in droplets, withprobes, amplification primers, and a sink aligned with their respectivebinding sites in wild-type (WT) and mutant (MUT) templates, inaccordance with aspects of the present disclosure.

FIG. 26 is a series of scatter plots of fluorescence data collected froma multiplex assay of a K-Ras target performed in droplets according tothe strategy of FIG. 25, with two different amounts of the sink presentin the droplets, in accordance with aspects of the present disclosure.

FIG. 27 is a schematic of a strategy for performing a duplex,allele-specific assay of target sequences, in accordance with aspects ofthe present disclosure.

FIG. 28 is a schematic view of a pair of exemplary reporters eachcomposed of base-pairing oligomers (a “probe” and a “primer”) thatcollectively provide a photoluminophore and an energy transfer partner,such as a quencher, for the photoluminophore, in accordance with aspectsof the present disclosure.

FIG. 29 is a schematic view of exemplary configurations produced byamplification of target in the presence of the reporters of FIG. 28, inaccordance with aspects of the present disclosure.

FIGS. 30A and 30B are a pair of scatter plots of fluorescence datacollected from a multiplex assay using two reporters, such as thereporters of FIG. 28, one reporter having a probe labeled with a FAMfluorophore and the other reporter having a probe labeled with a HEXreporter. FIG. 30A shows results from a “positive” sample containingtarget DNA. FIG. 30B shows results from a “negative” or no-targetsample.

FIG. 31 is a schematic scatter plot of fluorescence data illustratinghow the reporters of FIG. 28 may be used to design multiplex assays byallowing signal (open clusters) from the positive droplets to occupy aspace in 360 degrees around the signal (shaded cluster) for negativesample.

DETAILED DESCRIPTION

The present disclosure provides a system, including methods, apparatus,and compositions, for performing amplification assays with anamplification reporter including a first oligomer and a second oligomercapable of base-pairing with one another below a melting temperature ofthe reporter. The reporter may have a detectable photoluminescence thatis affected, such as reduced, by base-pairing of the first and secondoligomers with one another. A target, such as a nucleic acid targetsequence, may be amplified in at least one volume, such as a pluralityof partitions, above the melting temperature, and photoluminescence ofthe reporter may be detected from the at least one volume below themelting temperature. A property of the target, such as a concentrationof the target, may be determined based on the photoluminescencedetected.

The present disclosure enables lowering the photoluminescent backgroundof partitions that do not contain the product of interest (negativepartitions), to increase the difference in magnitude of the signals fornegative and positive partitions. A reporter composed of a pair ofbase-pairing oligomers may be included in the partitions. The reportermay include an energy donor and an energy acceptor that form aproximity-dependent energy transfer pair. The energy donor may beincluded in one of the oligomers and the energy acceptor in the other,or both may be included in the same oligomer. One of the oligomers, afirst (or second) oligomer, may be a “probe” that emits light to bedetected. The other of the oligomers, a second (or first) oligomer, maybe described as a “sink.” The sink affects (e.g., decreases, increases,and/or spectrally shifts, among others) a photoluminescence detectablefrom the probe/reporter when the probe and the sink are base-paired withone another, relative to when the probe is not base-paired with thesink. For example, the probe may include a photoluminophore that acts asan energy donor, and the sink may include a quencher that acts as anenergy acceptor for the photoluminophore, to reduce the basalphotoluminescence of the probe in the partitions.

The sink may include an oligonucleotide that is at least partially orfully complementary to the probe, allowing the sink to bind to theprobe. The oligonucleotide may, for example, bind to the probe topromote intramolecular and/or intermolecular quenching of thephotoluminophore by the quencher. In some examples, the probe may or maynot include a quencher, and the sink may include a quencher that reducesphotoluminescence emission by the photoluminophore when the sink bindsthe probe. In any event, a quencher of the probe and/or the sink may, insome cases, quench the photoluminophore of the probe by contactquenching (interchangeably termed static quenching).

In some embodiments, the sink may be configured to bind the probe duringonly a portion of an assay, based on the thermal profile of the assay.The sink may bind an intact form of the probe at a lower, detectiontemperature but not substantially at one or more higher, amplificationtemperatures. The detection temperature (also termed a readingtemperature) may be less than about 50, 45, 40, 35, or 30 degreesCelsius, or no more than about room temperature (about 20-25 degreesCelsius)), among others. The sink may not bind substantially to theprobe at one or more (or each) amplification temperatures (e.g., anannealing temperature and/or an extension temperature) and/or may notbind substantially to the probe at a minimum temperature used foramplification/thermocycling. Accordingly, the sink may increasequenching of the photoluminophore during detection, without interferingsubstantially with amplification, thus lowering the detectedphotoluminescence amplitude of negative partitions. The sink can be easyto design and can be added conveniently to an assay mixture. In otherexamples, the sink may be configured to bind to a probe during and/orafter each amplification cycle, to permit a kinetic analysis ofamplification (e.g., a real-time assay) in at least one fluid volume.

The present disclosure may provide a method to differentiate between asuccessful PCR reaction and an unsuccessful PCR reaction by detectinglight at a reading temperature. A probe and a complementary oligomer (asink) may be mixed with amplification reagents before thermal cycling.The sink may affect the photoluminescence of the probe at the readingtemperature, but may not affect the photoluminescence of the probeduring thermal cycling, whether or not amplification occurs. The sinkmay have a melting temperature for base-pairing with the probe that isbelow standard thermal-cycling conditions, but greater than the readingtemperature. The probe and the sink may interact at the readingtemperature to provide quenching in the absence of amplification. In thepresence of amplification, the sink may interact less with the probe dueto competition with an amplicon for binding to the probe, not due todisplacement of the sink from the probe during amplification.

Further aspects of the present disclosure are presented in the followingsections: (I) overview of reporters composed of base-pairing oligomers,(II) assays, (III) compositions, and (IV) examples.

I. OVERVIEW OF REPORTERS COMPOSED OF BASE-PAIRING OLIGOMERS

This section provides an overview of reporters composed of base-pairingoligomers and illustrates use of an exemplary reporter in anamplification assay to lower the background signal; see FIGS. 1-3.

FIG. 1 shows an exemplary reporter 50 having a base-paired configurationformed by binding of a probe 52 to a sink 54 via a series of base pairs56. The base pairs are intended to be schematic and may not (or may)represent the actual number of base pairs formed, here or elsewhere inthe present disclosure. The reporter interchangeably may be described asa reporter system and may have the base-paired form shown in FIG. 1, andat least one separated form. A separated form can be created by melting(also termed denaturing) the base-paired form. Other separated forms canbe created, at least in part, by modifying the probe and/or the sink,such as by extension or degradation.

The probe and the sink each may be a different oligomer. The oligomermay have a body formed by a chain (e.g., an oligonucleotide) ofconjugated units, such as nucleotides or nucleotide analogues, eachcontaining a base (e.g., a nucleobase). The body of each oligomer may beattached to at least one energy donor and/or at least one energyacceptor of an energy transfer pair, as described below.

Probe 52 may include a body formed by an oligonucleotide 58 (or anoligonucleotide analogue), at least one energy donor, such as aphotoluminophore 60, and, optionally, at least one energy acceptor, suchas a quencher (e.g., quencher 62 a). (In some examples, the quencher maybe omitted from the probe (e.g., see Section IV).) The photoluminophoreand the quencher each may be connected (e.g., conjugated,interchangeably termed covalently linked) to oligonucleotide 58 at anysuitable position along the oligonucleotide, such as at respectiveopposite ends of the oligonucleotide from each other. For example, inthe depicted embodiment, photoluminophore 60 is attached to the 5′-endand quencher 62 a is attached to the 3′-end of oligonucleotide 58. Inother cases, at least one quencher may be positioned internally, betweenthe 5′- and 3′-ends of the oligonucleotide (e.g., as in a ZEN™ probefrom Integrated DNA Technologies).

Probe 52 may or may not act as a primer for target amplification. If theprobe acts as a primer, the probe may be configured to be extendedduring amplification through the action of a polymerase (and/or ligase),and may define one of the ends of an amplicon resulting from targetamplification. For example, the probe may be a forward primer or areverse primer of a pair of primers for target amplification. (The terms“forward” and “reverse” for primers are arbitrary, interchangeabledesignations, unless specified otherwise.) If the probe is not intendedto act as a primer, the probe may be modified to block its elongationand/or may have one or more mismatches with the template at or near its3′-end, to discourage extension by polymerase (or ligase). Accordingly,the probe may bind to the end of a target/amplicon, or may bind at aposition that is intermediate the binding sites for a forward primer anda reverse primer used for target amplification.

Photoluminophore 60 may be any moiety capable of emitting light(photoluminescing) in response to irradiation with excitation light.Exemplary forms of photoluminescence include fluorescence andphosphorescence, among others. Accordingly, the photoluminophore may,for example, be a fluorophore or a phosphor. Suitable photoluminophoresmay include a dye, such as a FAM, VIC, HEX, ROX, TAMRA, or JOE dye, orthe like. In some examples, the photoluminophore may be replaced with aluminophore capable of chemiluminescence or any other form ofluminescence.

Sink 54 may include a body formed by a sink oligonucleotide 64 (or anoligonucleotide analogue) that is at least substantially complementaryto probe oligonucleotide 58 and binds specifically to probe 52, at leastin part via base pairs 56. The sink may (or may not) include at leastone energy donor and/or at least one energy acceptor that form an energytransfer pair with one another. For example, the sink may include atleast one quencher (e.g., quencher 62 b) connected (e.g., conjugated) tosink oligonucleotide 64 at any suitable position along theoligonucleotide. Each donor/acceptor of sink 54 may be connected at a3′-end of oligonucleotide 64, as shown, at a 5′-end, or intermediate the3′- and 5′-ends. Quencher 62 b may be positioned very close tophotoluminophore 60 in reporter 50, such as to permit contact quenching.For example, quencher 62 b and photoluminophore 60 may be conjugated torespective nucleotides that base-pair with one another (i.e., thenucleotides are aligned with one another), or that are offset by no morethan two nucleotides or no more than one nucleotide along thebase-paired reporter.

Each of oligonucleotides 58 and 64 may have any suitable properties. Theoligonucleotide may have any suitable length, such as at least 6, 8, 10,15, or 20 nucleotides, and/or less than about 200, 100, or 50nucleotides, among others. The oligonucleotide may be a conventionaloligonucleotide or an analogue or derivative thereof. Exemplaryanalogues/derivatives include peptide nucleic acids, locked nucleicacids, phosphorothiates, etc. Oligonucleotides 58 and 64 may have thesame length or different lengths. If the oligonucleotides have differentlengths, oligonucleotide 64 may be shorter (or longer) thanoligonucleotide 58. In some cases, a reporter with sink oligonucleotide64 shorter than probe oligonucleotide 58 may be preferable, because theprobe then can be designed to bind to the target/amplicon, but not thesink, at the annealing/extension temperature(s) used for amplification.Also or alternatively, the probe may be configured to bind to thetemplate/amplicon with a higher melting temperature than binding to thesink. The oligonucleotides may be configured to form any suitable numberof base pairs with one another, such as at least 5, 10, or 15 amongothers. A base-paired form of the reporter resulting from base pairingof the oligonucleotides with one another may have a melting temperature,in the assay, that is less than the average, maximum, or minimumtemperature used for amplification (e.g., less than theannealing/extension temperature(s) used for thermocycling), such as lessthan about 70, 60, or 50 degrees, among others. In some cases, the probemay be unquenched at the temperatures used for amplification, such thatall of the partitions appear to be positive for amplification ifphotoluminescence is detected from the partitions at an amplificationtemperature. Oligonucleotide 64 (and sink 54) may be configured to benon-extendable (or extendable) and non-cleavable (or cleavable) bypolymerase during amplification. For example, 3′ phosphorylation oranother modification can be used to prevent the probe (and/or sink) frombeing extended by polymerase (or ligase) during amplification.

Quenchers 62 a and 62 b, interchangeably termed quencher moieties, mayhave any suitable properties. Each quencher may be configured to reducean ability of photoluminophore 60 to emit light when excited (e.g., whenirradiated with suitable excitation light). If more than one quencher ispresent in a reporter, a probe, and/or a sink, the quenchers may becopies of one another or may be structurally distinct. A quencher may bea non-photoluminescent or “dark quencher” that is capable of quenchingemission of light from the photoluminophore without substantiallyemitting light itself, or may be a photoluminescent quencher that emitslight as a result of energy transfer (e.g., fluorescence resonanceenergy transfer) from the photoluminophore. Exemplary quenchers that maybe suitable include Black Hole® quenchers (BHQ), Iowa Black® darkquenchers (IB), TAMRA dye, and/or the like.

FIG. 2 shows a schematic comparing the levels of fluorescence that maybe produced in an assay by a base-paired form of reporter 50, probe 52separate from sink 54, and a cut form 70 of probe 52 (interchangeablytermed a degraded or cleaved probe/reporter). Cut probe 70 may, forexample, be formed by amplification of a target, through probe cleavagecatalyzed by a polymerase (e.g., Taq DNA polymerase). The lowest levelof fluorescence in the schematic is produced by reporter 50, becausephotoluminophore 60 is quenched by quencher 62 a of the probe and/orquencher 62 b of sink 54. If sink 54 is omitted from the assay, thelevel of fluorescence from intact probe 52 is higher than in thepresence of the sink. Accordingly, the background level of fluorescenceis higher. The highest level of fluorescence is produced by degradedprobe 70 because photoluminophore 60 is no longer connected to probequencher 62 a (and/or proximate sink quencher 62 b). More particularly,because probe oligonucleotide 58 is degraded, photoluminophore 60 andquencher 62 a are no longer covalently linked to one another, and sink54, if present in the assay, cannot bind stably to the degraded probe.

FIG. 3 shows a schematic flowchart illustrating selected steps andconfigurations of an exemplary assay performed in partitions withreporter 50 (i.e., probe 52 and sink 54). The configurations shown heremay be produced in a partition that contains at least one copy of atemplate 80 containing a target 82. The template may be double-stranded,with strands 84 and 86, as shown, or single-stranded. A forward primer88 (“F”), a reverse primer 90 (“R”), and probe 52 are shown at the topof FIG. 3, aligned with corresponding regions of template 80/target 82at which each can bind (after the template/target is denatured). Asecond, unbound copy of probe 52 is shown as dashed because sink 54 maybe in molar excess relative to the probe, indicated by an unbound copyof the sink at 91, such that substantially all of the probe is bound tothe sink at room temperature, before amplification has begun.

A first cycle of target amplification may be initiated by heatingtemplate 80 above its denaturation temperature. Annealing then may beperformed at a lower temperature that permits the forward primer andprobe 52, and the reverse primer, to bind specifically to single strands86 and 84 of the template. Probe 52 and sink 54 of reporter 50 mayremain separate from one another (i.e., not base-paired) throughoutamplification, if amplification is performed above the meltingtemperature of the reporter. Accordingly, sink 54 may not bind to probe52 until after amplification has been completed.

Each of the forward and reverse primers then may be extended during thefirst cycle to produce primer extension products 92, 94 bound torespective strands 86 and 84 of template 80. During extension of theforward primer, the polymerase encounters probe 52 bound to strand 86and catalyzes cleavage of probe oligonucleotide 58, to form degradedprobe 70.

Additional cycles of amplification may be performed to generate amplicon96 (interchangeably termed an amplified target), until an endpoint ofamplification is reached. Each copy of amplicon 96 may have boundariesdefined by primers 88 and 90, or other primers if, for example, nestedamplification is performed. In any event, degraded probe 70 accumulatesduring amplification, while intact probe 52 is depleted. Accordingly,the amplitude (interchangeably termed the magnitude or the level) of thephotoluminescence detectable from (and/or or dependent on)photoluminophore 60 is higher if at least one copy of template 80/target82 is present in the partition before amplification. The difference inphotoluminescence amplitude between a negative partition and a positivepartition is increased because sink 54 is bound to probe 52 duringdetection if the probe is not degraded.

II. ASSAYS

This section describes exemplary amplification assays that may beperformed with a sink and an exemplary system for performing a digitalassay with a sink; see FIGS. 4 and 5.

FIG. 4 shows a flowchart of an exemplary method 110 of performing anassay or analysis. The steps presented for method 110 may be performedin any suitable order and in any suitable combination. Furthermore, thesteps may be combined with and/or modified by any other suitable steps,aspects, and/features of the present disclosure.

Volume Formation.

At least one volume may be prepared for assay of a target, indicated at112. The at least one volume may include a template corresponding to thetarget, a probe, and a sink. The volume also may include reagents foramplification of the target, such as one or more primers, anamplification enzyme (e.g., a polymerase or ligase), dNTPs/NTPs, and thelike. Further aspects of the composition of a reaction mixtureconstituting the at least one volume are described below in Section III.

Preparation of the reaction mixture may include or be described aspreparation of a sample. Preparation of the sample may include anysuitable manipulation of the sample, such as collection, dilution,concentration, purification, lyophilization, freezing, extraction,combination with one or more assay reagents, performance of at least onepreliminary reaction to prepare the sample for one or more reactions inthe assay, or any combination thereof, among others. Preparation of thesample may include rendering the sample competent for subsequentperformance of one or more reactions, such as one or more enzymecatalyzed reactions and/or binding reactions.

In some embodiments, preparation of the sample may include combining thesample with reagents for amplification and for reporting whether or notamplification occurred. Reagents for amplification may include anycombination of one or more primers for synthesis of an ampliconcorresponding to the target, dNTPs and/or NTPs, at least one enzyme(e.g., a polymerase, a ligase, a reverse transcriptase, a restrictionenzyme, or a combination thereof, each of which may or may not beheat-stable), and/or the like. Accordingly, preparation of the samplemay render the sample (or partitions thereof) capable of amplificationof each of one or more targets, if present, in the sample (or apartition thereof). Reagents for reporting may include a differentreporter for each target of interest. Accordingly, preparation of thesample for reporting may render the sample capable of reporting, orbeing analyzed for, whether or not amplification has occurred, on atarget-by-target basis, and optionally the extent of any suchamplification.

Formation of the reaction mixture may include forming a continuous phaseor bulk phase containing all of the components necessary for targetamplification. Alternatively, or in addition, formation of the reactionmixture may include fusing partitions, such as droplets (see below).

The term “luminescence” means emission of light that cannot beattributed merely to the temperature of the emitting body. Exemplaryforms of luminescence include photoluminescence, chemiluminescence,electroluminescence, or the like. A “luminophore” is any atom,associated group of atoms, moiety, molecule, or associated group ofmolecules capable of luminescence. Photoluminescence is any luminescenceproduced in response to irradiation with excitation light and includesfluorescence, phosphorescence, etc. Accordingly, a luminophore may be aphotoluminophore, such as a fluorophore or a phosphor, among others.

A target interchangeably may be termed an analyte, a species, or, insome cases, a template.

Partition Formation.

Partitions may be formed each containing a portion of the same reactionmixture and/or sample, and/or each containing a portion of an incompleteprecursor to the reaction mixture (if the partitions are to besupplemented with one or more additional reaction components). Eachtarget may be amplified from a template, such as from a single copy ofthe template if present in a given partition.

The partitions when provided (e.g., when formed) may contain a target at“partial occupancy,” which means that one or more of the partitions donot contain at least one copy of the target. In other words, only asubset of the partitions contains at least one copy of the target.Accordingly, with partial occupancy, one or more (e.g., a plurality) ofthe partitions contain no copies of the target, one or more (e.g., aplurality) of the partitions may contain a single copy (only one copy)of the target, and, optionally, yet one or more of the partitions (e.g.,the rest of the partitions) may contain two or more copies of thetarget.

The term “partial occupancy” is not restricted to the case where thereis no more than one copy of a particular template/target of interest inany partition. Partitions containing a template and/or a target atpartial occupancy may, for example, contain an average of more than, orless than, about one copy, two copies, or three copies, among others, ofthe template/target per partition when the partitions are provided orformed. Copies of a template (and/or target) may have a randomdistribution among the partitions, which may be described as a Poissondistribution.

Partition formation may involve distributing any suitable portionincluding up to all of the sample/reaction mixture to the partitions.Each partition may be and/or include a fluid volume that is isolatedfrom fluid volumes of other partitions. The partitions may be isolatedfrom one another by a fluid/liquid phase, such as a continuous phase ofan emulsion, by a solid phase, such as at least one wall of a container,or a combination thereof, among others. In some embodiments, thepartitions may be droplets disposed in a continuous phase, such that thedroplets and the continuous phase collectively form an emulsion.

The partitions may be formed by any suitable procedure, in any suitablemanner, and with any suitable properties. For example, the partitionsmay be formed with a fluid dispenser, such as a pipette, with at leastone droplet generator having an orifice and/or a channel intersection atwhich droplets are created, by agitation of the sample/reaction mixture(e.g., shaking, stirring, sonication, etc.), and/or the like.Accordingly, the partitions may be formed serially, in parallel, or inbatch. The partitions may have any suitable volume or volumes. Thepartitions may be of substantially uniform volume or may have differentvolumes. Exemplary partitions having substantially the same volume aremonodisperse droplets. Exemplary volumes for the partitions include anaverage volume of less than about 100, 10 or 1 μL, less than about 100,10, or 1 nL, or less than about 100, 10, or 1 pL, among others.

Partitions competent for amplification of each target may be formeddirectly from a bulk phase containing the template, or may be formed inmultiple steps. In some cases, the step of forming partitions mayinclude dividing a bulk phase into isolated fluid volumes (such asdroplets) containing the template at partial occupancy. The fluidvolumes may be the partitions themselves or may contribute to thepartitions. For example, the fluid volumes may be a first set of fluidvolumes, and the step of forming partitions may include combiningindividual fluid volumes of the first set with individual fluid volumesof a second set. The second set may include one or more reagents foramplification of one or more of the targets, such as at least one primerfor amplification of at least one of the targets, a probe, a sink, orthe like. The step of combining may include fusing fluid volumes of thefirst set individually with fluid volumes of the second set, such asfusing droplets containing the template with droplets containing primersfor amplification of one or more targets from the template.

Target Amplification.

Each target may be amplified, indicated at 114. Amplification may beperformed in partitions (a dispersed phase) or in a continuous phase,such as in the reaction mixture without forming partitions. If performedin partitions, amplification of each target may occur selectively(and/or substantially) in only a subset of the partitions, such as lessthan about three-fourths, one-half, one-fourth, or one-tenth of thepartitions, among others. Amplification of each target may occurselectively or exclusively in partitions containing at least one copy ofthe target (i.e., containing at least one copy of a templatecorresponding to the target).

Amplification may or may not be performed isothermally. In some cases,amplification in the partitions (and/or at least one volume) may beencouraged by heating the partitions (and/or volume) and/or incubatingthe partitions (and/or volume) at a temperature above room temperature,such as at a denaturation temperature (e.g., greater than about 90degrees Celsius), an annealing temperature (e.g., about 50-75 degreesCelsius), and/or an extension temperature (e.g., about 60 to 80 degreesCelsius), for one or a plurality of cycles. In some examples, thepartitions (and/or volume) may be thermally cycled to promoteamplification by a polymerase chain reaction and/or ligase chainreaction, among others. Exemplary isothermal amplification approachesthat may be suitable include nucleic acid sequence-based amplification,transcription-mediated amplification, multiple-displacementamplification, strand-displacement amplification, rolling-circleamplification, loop-mediated amplification of DNA, helicase-dependentamplification, and single-primer amplification, among others.

Light Detection.

Photoluminescence of the reporter may be detected, optionally below thereporter melting temperature, indicated at 116. Light detected may beemitted by the photoluminophore of the reporter directly or may beemitted by an energy transfer partner of the photoluminophore, amongothers. Detection of light may be described as collection ofamplification data. The data may be collected by detecting light emittedfrom individual partitions or from the at least one volume. The lightmay be emitted in response to irradiation of the partitions or volumewith excitation light for the photoluminophore. The data may becollected for emission of light from the partitions, or volume, in onewavelength/waveband (one optical channel), a pair ofwavelengths/wavebands (two optical channels), or the like.

An optical channel may represent a particular detection regime withwhich emitted light is generated and detected. The detection regime maybe characterized by a wavelength/waveband (i.e., a wavelength regime)for detection of emitted light. If pulsed excitation light is used inthe detection regime to induce light emission, the detection regime maybe characterized by a wavelength or waveband for illumination withexcitation light and/or a time interval during which light emission isdetected with respect to each light pulse. Accordingly, optical channelsthat are different from each other may differ with respect to thewavelength/waveband of excitation light, with respect to thewavelength/waveband of emitted light that is detected, and/or withrespect to the time interval during which emitted light is detectedrelative to each pulse of excitation light, among others.

Data collection may include generating one or more signalsrepresentative of light detected from individual partitions or thereaction mixture. The signals may represent an aspect of light, such asthe intensity, polarization, or lifetime of the light, among others. Thesignals optionally may include data collected in two or more differentoptical channels (e.g., in different wavelengths/wavelength ranges(wavebands) and/or color regimes) from probes/reporters for the sameand/or different targets). The light detected from each probe/reportermay be light emitted by a photoluminophore (e.g., a fluorophore). Thelight detected in a given channel may be detected such that light fromdifferent probes/reporters is summed or accumulated without attributionto a particular probe/reporter. Thus, the signal for a given channel maybe a composite signal that represents two, three, four, or more assaysand thus two, three, four, or more targets. In other cases, the signalsfor the targets may be detected in different optical channels.

The signal(s) may be created based on detected light emitted from one ormore probes/reporters in the partitions. The one or moreprobes/reporters may report whether at least one of two or moreparticular amplification reactions represented by the signal hasoccurred in a partition and thus whether at least one copy of at leastone of two or more particular targets corresponding to the two or moreparticular amplification reactions is present in the partition. Thelevel or amplitude of the signal corresponding to the reporters may beanalyzed to determine whether or not at least one of the particularamplification reactions has occurred and at least one copy of one of theparticular targets is present. The level or amplitude of the signal mayvary among the partitions according to whether at least one of theparticular amplification reactions occurred or did not occur and atleast one of the particular targets is present or absent in eachpartition. For example, a partition testing positive for a particulartarget only may produce a signal value that is above a given thresholdand/or within a given range. Partitions may be analyzed and signalscreated at any suitable time(s). Exemplary times include at the end of areaction phase of the assay (an endpoint assay), when reactions have runto completion and the data no longer are changing, or at some earliertime, as long as the data are sufficiently and reliably separated.

The reporters may have any suitable structure and characteristics. Eachreporter may be a probe including an oligonucleotide and aphotoluminophore associated with the oligonucleotide (e.g., with thephotoluminophore covalently attached to the oligonucleotide), to labelthe oligonucleotide. The probe also may or may not include an energytransfer partner for the photoluminophore, such as a quencher or anotherphotoluminophore. The probe may be capable of binding specifically to anamplicon (e.g., a strand thereof) produced by amplification of a target.The probe may or may not also function as an amplification primer thatforms part of an amplicon in the assay. Exemplary labeled probes includeTaqMan@ probes, Scorpion® probes/primers, Eclipse® probes, Amplifluor®probes, molecular beacon probes, Lux® primers, proximity-dependent pairsof hybridization probes that exhibit FRET when bound adjacent oneanother on an amplicon, QZyme® primers, or the like.

In some cases, at least one of the reporters may be a generic reporter,such as a dye, that binds nucleic acid relatively nonspecifically. Forexample, the dye may have no covalent attachment to an oligonucleotidethat confers substantial sequence binding specificity. The dye may be amajor groove binder, a minor groove binder, an intercalator, or anexternal binder, among others. The dye may bind preferentially todouble-stranded relative to single-stranded nucleic acid and/or mayexhibit a greater change in a photoluminescent characteristic (e.g.,intensity) when bound to double-stranded relative to single-strandednucleic acid. Exemplary dyes that may be suitable include luminescentcyanines, phenanthridines, acridines, indoles, imidazoles, and the like,such as DAPI, Hoechst® 33258 dye, acridine orange, etc. Exemplaryintercalating dyes that may be suitable include ethidium bromide,propidium iodide, EvaGreen® dye, SYBR® Green dye, SYBR® Gold dye, and7-aminoactinomycin D (7-AAD), among others. Multiplexed assay of two ormore targets may be performed in the same partitions with two or moredistinct target-specific probes and/or at least one target-specificprobe and a generic reporter.

Population Identification.

Partition populations (interchangeably termed clusters or bands) thattest negative or positive for one or more targets may be identified fromthe data. Identification may be performed by a data processor using analgorithm (e.g., an algorithm that identifies patterns (e.g., partitionclusters) in the data), by a user, or a combination thereof. In somecases, a data processor may produce and output (e.g., display) a plot ofthe collected data (e.g., a graph, a 2-D scatter plot, a histogram, orthe like). The user then may define the boundary of each populationbased on the plot(s), e.g., through a graphical user interface to definepopulation boundaries, or by inputting values (e.g., representingamplitude thresholds/ranges) to define a boundary for each population.Each population boundary may be defined by one or more ranges of values,a geometrical shape that surrounds the population (e.g., a polygon,ellipse, etc.), an algorithm, or the like. Accordingly, the populationboundary may be determined by the user, automatically by a processorthrough an algorithm, or a combination thereof.

Identification of partition populations may include assigning eachpartition to one of a plurality of predefined bins each corresponding toa distinct partition population. The predefined bins may represent allcombinations of negatives and positives for the targets.

Obtaining Partition Counts.

A partition count for each partition population may be obtained. Thepartition count may be a value representing the number of partitionsconstituting a particular partition population.

Determination of Target Property.

A property of at least one target may be determined based on thephotoluminescence detected (i.e., on the collected data), indicated at118. The property may be a level (e.g., a concentration), activity,configuration, location, or the like. The level may represent the levelof target/template that was present before amplification. Determinationof target levels may (or may not) be based on each target having aPoisson distribution among the partitions. Each level may, for example,be a value representing the total number of partitions positive (ornegative) for the target/template/amplicon, or a concentration value,such as a value representing the average number of copies of thetarget/template per partition or unit volume, among others. Thepartition data further may be used (e.g., directly and/or asconcentration data) to estimate copy number (CN) and copy numbervariation (CNV), or any other property of the sample, using any suitablealgorithms.

A level (e.g., concentration) of each target may be determined withPoisson statistics. The concentration may be expressed with respect tothe partitions (or reaction mixture) and/or with respect to a sampleproviding the target. The concentration of the target in the partitionsmay be calculated from the fraction of positive partitions (or,equivalently, the fraction of negative partitions) by assuming thatcopies of the target (before amplification) have a Poisson distributionamong the partitions. With this assumption, the fraction f(k) ofpartitions having k copies of the template is given by the followingequation:

$\begin{matrix}{{f(k)} = {\frac{\lambda^{k}}{k!}e^{- \lambda}}} & (1)\end{matrix}$

Here, λ is the concentration of the target in the partitions, expressedas the average number of target copies per partition (beforeamplification). Simplified Poisson equations may be derived from themore general equation above and may be used to determine targetconcentration from the fraction of positive partitions. An exemplaryPoisson equation that may be used is as follows:

$\begin{matrix}{\lambda = {{- \ln}\; \left( {1 - \frac{N_{+}}{N_{tot}}} \right)}} & (2)\end{matrix}$

where N₊ is the number of partitions (i.e., the partition count)positive for a given target, and where N_(tot) is the total number ofpartitions that are positive or negative for the target. N_(tot) isequal to a sum of (a) N₊ for the target and (b) the number of partitionsnegative for the target, or N⁻. N₊/N_(tot) (or N₊/(N₊+N⁻)) is equal tof₊, which is the fraction of partitions positive for the template (i.e.,f₊=f(1)+f(2)+f(3)+ . . . ) (see Equation 1), and which is a measuredestimate of the probability of a partition having at least one copy ofthe template. Another exemplary Poisson equation that may be used is asfollows:

$\begin{matrix}{\lambda = {{- \ln}\; \left( \frac{N_{-}}{N_{tot}} \right)}} & (3)\end{matrix}$

where N⁻ and N_(tot) are as defined above. N⁻/N_(tot) is equal to f⁻,the fraction of negative partitions (or 1−f₊), and is a measuredestimate of the probability of a partition having no copies of thetarget, and A is the target concentration as described above.

Equations 2 and 3 above can be rearranged to produce the following:

λ=ln(N _(tot))−ln(N _(tot) −N ₊)  (4)

λ=ln(N _(tot))−ln(N ⁻)  (5)

The concentration of each target in an assay can, for example, bedetermined with any of Equations 2 to 5, using values (i.e., partitioncounts) obtained for N_(tot) and N⁻ or, equivalently, N₊, for eachtarget. In some cases, the value used for N_(tot) (the total partitioncount) may be the same for each target. In other cases, the value usedfor N_(tot) may vary, such as if some of the populations are excludedfrom the total count due to population overlap. In some embodiments,N_(tot) may be equivalent to a combination of all populations, namely, asum of the partition counts for all populations identified.

In some embodiments, an estimate of the level of a target (and/or thetemplate) may be obtained directly from the positive fraction, withoutuse of Poisson statistics. In particular, the positive fraction and theconcentration (copies per partition) converge as the concentrationdecreases. For example, with a positive fraction of 0.1, theconcentration is determined with Equation 2 to be about 0.105, adifference of only 5%; with a positive fraction of 0.01, theconcentration is determined to be about 0.01005, a ten-fold smallerdifference of only 0.5%. However, the use of Poisson statistics canprovide a more accurate estimate of concentration, particularly with arelatively higher positive fraction, because Poisson statistics takesinto account the occurrence of multiple copies of the sametarget/template in the same partition.

Further aspects of sample preparation, partition formation, datacollection, population identification, obtaining partition counts, andtarget level determination, among others, that may be suitable for thesystem of the present disclosure are described elsewhere in the presentdisclosure, and in the references identified above in theCross-References, which are incorporated herein by reference.

FIG. 5 shows an exemplary system 120 for performing the assay of FIG. 4.System 120 may include a partitioning assembly, such as a dropletgenerator 122 (“DG”), a thermal incubation assembly, such as athermocycler 124 (“TC”), a detection assembly (a detector) 126 (“DET”),and a data processing assembly (a data processor) 128 (“PROC”), or anycombination thereof, among others. The data processing assembly may be,or may be included in, a controller that communicates with and controlsoperation of any suitable combination of the assemblies. The arrowsbetween the assemblies indicate movement or transfer of material, suchas fluid (e.g., a continuous phase of an emulsion) and/or partitions(e.g., droplets) or signals/data, between the assemblies. Any suitablecombination of the assemblies may be operatively connected to oneanother, and/or one or more of the assemblies may be unconnected to theother assemblies, such that, for example, material/data are transferredmanually.

Detector 126 may provide a single channel or a plurality of opticalchannels in which data can be collected. The detector may have adistinct sensor or detection unit for each optical channel. The detectormay be operatively associated with a light source configured to exciteone or more photoluminophores of the assay.

System 120 may operate as follows. Droplet generator 122 may formdroplets disposed in a continuous phase. The droplets may be cycledthermally with thermocycler 124 to promote amplification of one or moretargets in the droplets. Signals may be detected from the droplets withdetector 126. The signals may be processed by processor 128 to determineone or more droplet counts and/or target levels, among others.

III. COMPOSITIONS

This section provides exemplary compositions of the present disclosure.Each composition may or may not contain all the reagents necessary foramplification of a nucleic acid target.

The composition may be a continuous phase (interchangeably termed a bulkphase) or may be composed of partitions that are isolated from oneanother. If composed of partitions, the partitions may be aqueouspartitions separated from one another by a solid phase (e.g., at leastone wall of at least one container) and/or by a liquid phase, amongothers. The liquid phase may be a nonaqueous continuous phase thatsurrounds each of the partitions. The continuous phase may include atleast one surfactant.

The nonaqueous phase may serve as a carrier fluid forming a continuousphase that is immiscible with water. The nonaqueous phase may be an oilphase comprising at least one oil, but may include any liquid (orliquefiable) compound or mixture of liquid compounds that is immisciblewith water. The oil may be synthetic or naturally occurring. The oil mayor may not include carbon and/or silicon, and may or may not includehydrogen and/or fluorine. The oil is hydrophobic and may be lipophilicor lipophobic. In other words, the oil may be generally miscible orimmiscible with organic solvents. Exemplary oils may include at leastone silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or acombination thereof, among others.

In exemplary embodiments, the oil is a fluorinated oil, such as afluorocarbon oil, which may be a perfluorinated organic solvent. Afluorinated oil may be a base (primary) oil or an additive to a baseoil, among others. Exemplary fluorinated oils that may be suitable aresold under the trade name FLUORINERT (3M), including, in particular,FLUORINERT Electronic Liquid FC-3283, FC-40, FC-43, and FC-70. Anotherexample of an appropriate fluorinated oil is sold under the trade nameNOVEC (3M), including NOVEC HFE 7500 Engineered Fluid.

The composition may comprise any suitable combination of a template, atleast one probe, at least one sink, one or more primers (at least one ofwhich also may be a probe or a sink), dNTPs and/or NTPs, a polymerase(e.g., a heat stable polymerase), a ligase, a reverse transcriptase,water, buffer, and a carrier fluid that is immiscible with water, amongothers.

The template may be a nucleic acid, such as DNA and/or RNA, and may beat least predominantly single-stranded or double-stranded, among others.The template may be fragmented to any suitable size or size range.

The probe may include any combination of an oligonucleotide, at leastone photoluminophore and at least one quencher for the photoluminophore.The oligonucleotide may be complementary to a region of an amplicon suchthat the oligonucleotide can bind to the amplicon during targetamplification that generates the amplicon. The region of the ampliconmay or may not be present in the template/target from which the ampliconis generated. Each of the photoluminophore and the quencher may beconjugated to the oligonucleotide. The quencher may be positioned (inthe absence of a sink) for dynamic quenching and/or contact quenching.

The sink may bind the probe to affect an ability of at least onephotoluminophore of the probe to emit light. For example, the sink mayfunction to bring an energy transfer partner closer to thephotoluminophore of the probe. The sink may include an oligonucleotidethat is complementary to the probe, and also may include at least oneenergy transfer partner (such as a quencher) for the photoluminophore.Accordingly, the sink may bind the probe to position an energy transferpartner of the sink closer to the photoluminophore of the probe, and/orto position an energy transfer partner of the probe closer to thephotoluminophore of the probe. If the sink lacks an energy transferpartner, the sink may have a 5′-portion that binds a 5′-portion of theprobe and may have a 3′-portion that binds a 3′-portion of the probe(e.g., see Example 5).

The one or more primers may be configured to prime synthesis of at leastone amplicon strand and/or complementary amplicon strands. The one ormore primers thus may bind to the template and/or the amplicon and maydefine the ends of the amplicon. In exemplary embodiments, the one ormore primers are a sense primer and a distinct antisense primer thatcollectively define the ends of the amplicon.

IV. EXAMPLES

This section describes selected aspects and embodiments of the presentdisclosure related to assays with a reporter including a sink. Anysuitable aspects of the assay and reporter configurations disclosed inthe section may be combined with one another and/or with aspects ofassay and reporter configurations disclosed elsewhere in the presentdisclosure. These examples are intended for illustration only and shouldnot limit or define the entire scope of the present disclosure.

Example 1. Sink for a Primer

This example describes an exemplary assay performed with a sink capableof binding to a probe that functions as a labeled amplification primer;see FIGS. 6, 7, and 7A.

FIG. 6 shows a schematic comparing levels of fluorescence in an assayperformed with an exemplary reporter 130 including a labeled primer(probe 52) that binds to a sink 54. Primer oligonucleotide 58 may (ormay not) have a 3′ priming region 132 that is dedicated to binding to atarget and which has no complementarity to oligonucleotide 64 of sink54. Priming region 132 (and/or primer oligonucleotide 58) can bind to atemplate/target at an annealing temperature to permitpolymerase-mediated primer extension during amplification. The primingregion may, for example, have a length of at least about 10, 15, or 20nucleotides, among others. Oligonucleotide 58 also may have a 5′sink-binding region 134 that is dedicated to binding to sink 54 andwhich forms hydrogen bonds with the, to position photoluminophore 60 ofsink-binding region 134 proximate to a quencher 62 at a lowertemperature used for detection. Accordingly, the background level offluorescence measured from the photoluminophore of reporter 130 may below. Sink-binding region 134 may, for example, have a length of at leastabout 5, 10, or 15 nucleotides, among others. In some examples, regions132 and 134 may overlap substantially or completely. Sink-binding region134 may or may not be complementary to a template containing the targetto be amplified.

Amplification of a target with probe 52 as a primer (and optionally atleast one other primer) may generate an amplicon 140. A strand 142 ofamplicon 140 binds to the extended probe and prevents sink 54 frombinding to the extended probe 52 at a lower temperature that may be usedfor detection, indicated at 144. Since quencher 62 is not held proximatephotoluminophore 60 during detection, the level of fluorescence measuredfrom the photoluminophore incorporated into amplicon 140 may berelatively high compared to the background level of fluorescence for thereporter.

In other words, the extended probe may preferentially bind to thelonger, complementary amplicon strand, due to the higher meltingtemperature of the resulting reporter, and not to the sink. Inpartitions where amplification does not occur, the non-extended probemay be bound at the detection temperature by the sink, which may quenchemission of light from the probe. Assays based on this approach may bereferred to as Amplicon Mediated Probe assays (AMP assays).

FIG. 7 shows selected steps and configurations of an exemplary assaythat may be performed in partitions with reporter 130 of FIG. 6. Theconfigurations shown here may be produced in a partition that containsat least one copy of template 80 containing target 82. The template maybe double-stranded with strands 84 and 86, as shown, or single-stranded.A forward primer (“F”) (i.e., probe 52 from reporter 130) and a reverseprimer (“R”), are shown at the top of FIG. 7, aligned with correspondingregions of template 80/target 82 at which each can bind (after thetemplate/target is denatured). Sink 54 may be in molar excess relativeto probe 52, such that substantially all of the probe is bound to thesink at room temperature before amplification (and unbound copies ofsink 54 may be present in the partition).

A first cycle of target amplification may be initiated by heatingtemplate 80 above its denaturation temperature. Annealing then may beperformed at a temperature that permits probe 52 (and particularlypriming region 132) to bind specifically to single strand 86 of thetemplate. Binding of the reverse primer to strand 84 also occurs duringthe first cycle but is not illustrated here to simplify thepresentation.

Each of the forward and reverse primers then may be extended during thefirst cycle to produce primer extension products bound to respectivestrands of template 80. Depicted extension product 92 may be generatedby elongation of the forward primer (probe 52) with strand 86 serving asa template.

A second cycle of target amplification may be performed to generate acomplete copy of amplicon 140 using extension product 92 as template.After denaturation, the reverse primer binds to extension product 92 andmay be elongated. Accordingly, an extension product 146 incorporatingthe reverse primer can be elongated to a position aligned with the5′-end of product 92, causing sink-binding region 134 of product 92 tobe unavailable for later binding to sink 54, when the temperature isreduced. Additional cycles of amplification may be performed to increasethe number of copies of amplicon 140 until an endpoint of amplificationis reached. Successful amplification causes a decrease in the amount ofquenched reporter 130 present during detection, generally in directrelation to the amount of photoluminophore-labeled amplicon 140generated.

FIG. 7A shows an exemplary thermal profile for stages of the assay ofFIGS. 6 and 7, and the status of reporter 130 (e.g., annealed,denatured, and/or covalently modified) during each stage. The stagesidentified on the horizontal axis of the graph are volume formation,thermocycling (and/or amplification), and detection. The thermal profiledescribed here and below may apply to any of the reporter and assayconfigurations of the present disclosure.

Volume formation generally includes formation of one or more volumes foruse in the amplification stage and/or detection stage. The formation ofat least one volume may include mixing amplification reagents and sampleand/or dividing a bulk volume including amplification reagents and/orsample into partitions (e.g., droplets). Volume formation may (or maynot) be performed below the melting temperature of the reporter, at onemore different temperatures. For example, a bulk volume may be formed bymixing a sample and reagents below room temperature (such as on ice).The bulk volume then may (or may not) be divided into partitions at roomtemperature (e.g., about 20-25 degrees Celsius) or below roomtemperature, among others. In any event, partitions may be formed belowthe melting temperature of the reporter, such that the reporter remainsin a base-paired form 150, during partition formation (and/or bulkvolume formation).

Amplification may be performed by incubating the at least one volume ata single temperature or two or more different temperatures above themelting temperature of the reporter. Accordingly, incubation may beisothermal or may include thermally cycling (i.e., thermocycling) the atleast one volume, through a plurality of thermal cycles (also calledamplification cycles), such as at least 5, 10, or 15 cycles, amongothers. The at least one volume may remain above the melting temperatureof the reporter during an annealing portion and/or a primer extensionportion of each amplification cycle. The reporter may be in a separatedform 152 during any suitable portion of each amplification cycle, suchas throughout each of a plurality of amplification cycles. At least aportion of the reporter, probe, and/or sink may be covalently modifiedby amplification, such as extended to form extension product 92 and/orcleaved to form a degradation product.

Detection of light from reporter 130 may be performed below the meltingtemperature of the reporter. A portion that the reporter that is notmodified during amplification may be present in base-paired form 150.Another portion of the reporter may be present in a separated formcreated by base-pairing of extension product 92 with complementaryextension product 146 of amplicon 140, with sink 54 left unpaired.

Example 2. Reporter with a Single Quencher

This example describes an exemplary reporter in which the probe containsno quencher, with the probe being degraded during target amplification;see FIG. 8.

FIG. 8 shows a schematic comparing levels of fluorescence in an assayperformed with a reporter 160 including a probe 52 that does not have acovalently linked quencher (compare with FIG. 1). Sink 54 provides aquencher 62, which may provide sufficient quenching as the only quenchermoiety in the reporter, to give a low fluorescence background signal.Degraded probe 70, generated as a byproduct of amplification, produces arelatively high fluorescence signal because quencher 62 of sink 54 is nolonger held proximate photoluminophore 60.

Example 3. Reporter with Sink Degradation

This example describes an exemplary reporter in which a probe is anamplification primer and contains no quencher, with a sink of thereporter being degraded during target amplification; see FIG. 9.

FIG. 9 shows a schematic comparing levels of fluorescence in an assayperformed with a more stable version of reporter 130 of FIG. 6 and apolymerase capable of catalyzing cleavage of sink 54 during targetamplification. Reporter 130 may, for example, be more stable than inFIG. 6 because sink 54 may be longer (compare FIGS. 6 and 9). Thereporter may be sufficiently stable to form during the annealing step ofan amplification cycle and to remain base-paired during the extensionstep of the cycle. Partitions lacking a copy of the target exhibit a lowlevel of background fluorescence because sink 54 of reporter 130 remainsintact. Partitions having at least one copy of the target exhibit arelatively high level of fluorescence; formation of amplicon 140 resultsin a degraded form 170 of sink 54 that cannot quench photoluminophore 60efficiently.

Example 4. Amplification Primer as a Sink

This example describes an exemplary reporter 180 in which sink 54 isconfigured as an amplification primer; see FIG. 9A.

Reporter 180 may be structured generally as described above for reporter130 of FIG. 6, except that the positions of a photoluminophore 60 and aquencher 62 are reversed. In particular, quencher 62 may be conjugatedto a primer oligonucleotide 182. Oligonucleotide 182 may have a primingregion 184 and a probe-binding region 186, each of which may bestructured as described above for counterpart regions of oligonucleotide58 of FIG. 6. (For example, probe-binding region 186 may be completelycontained in priming region 184.) Quencher 62 may be attached to eitherregion and at any suitable position along the region. A probe 52 of thereporter may bind region 186 of oligonucleotide 182. Probe providesphotoluminophore 60 and may or may not be complementary to the ampliconproduced by amplification. Quenching of photoluminophore 60 may bediminished if the probe is degraded during amplification (if reporter180 is stable enough to be present during amplification) and/or if theprobe is excluded from binding to extended sink 54 at a detectiontemperature by amplicon formation.

Example 5. Sink without a Quencher

This example describes exemplary reporters 200, 202 in which probe 52 isplaced in a quenched configuration by a sink 54 containing no quencher;see FIGS. 10 and 11.

Sink 54 may be structured as an oligonucleotide that is not conjugatedto a quencher (or a photoluminophore). The oligonucleotide may include a5′-portion 206 and a 3′-portion 208 that are respectively complementaryto and bind to a 5′-portion 210 and a 3′-portion 212 of probe 52. As aresult, a photoluminophore 60 and a quencher 62 of the probe are held inproximity to each other in the reporter, reducing the basal luminescenceof the probe, such as by contact quenching. Sink 54 may have a3′-phosphorylation or other modification or structure to preventextension of the sink, which could cause probe cleavage. The sink alsoor alternatively may be structured to be resistant topolymerase-mediated cleavage.

The length of probe 52 may determine whether the probe is preferentiallycircularized to form a circular hybrid of reporter 200, which isquenched intramolecularly, or arranged generally end-to-end in series,to form a chain hybrid of reporter 202 (interchangeably termed a linearhybrid), which is quenched intermolecularly. Circular hybrid 200 usesthe same copy of probe 52 to provide a copy of photoluminophore 60 and acopy of quencher 62 that quenches the photoluminophore copy. Chainhybrid 202 uses a pair of copies of probe 52 to provide a copy ofquencher 62 that quenches a copy of photoluminophore 60. The length of aspacer region 214 of probe 52 that extends from end region 210 to endregion 212 can determine whether the probe preferentially forms hybrid200 or 202. For example, if the spacer region is long enough, as in FIG.10, the same copy of the probe can bind to distinct regions of the samecopy of sink 54. If the spacer region is not long enough for probecircularization, the chain hybrid of FIG. 11 may be formed instead. Insome cases, spacer region 214 may be short, such as no more than four,three, two, or one nucleotide, to optimize the quenching of thephotoluminophore by the quencher (e.g., such that contact quenchingoccurs). Sink 54 also may have a spacer region 216 that connects endportions 206 and 208. Spacer region 216 may be kept short, such as nomore than four, three, two, or one nucleotide, to optimize quenching ofthe photoluminophore by the quencher (e.g., such that contact quenchingoccurs).

Example 6. Reporter with Nonterminal Photoluminophore and Quencher

This example describes an exemplary reporter 220 formed by a probe 52and a sink 54 providing a photoluminophore 60 and a quencher 62 atnonterminal positions along respective oligonucleotides of the probe andsink; see FIG. 12. In other embodiments, one member of an energytransfer pair may be located at a terminal position (conjugated to the5′- or 3′-end of an oligomer chain), and the other member of the energytransfer pair may be disposed at a nonterminal position of the sameoligomer chain or at a nonterminal position of an at least partiallycomplementary oligomer chain.

Example 7. Assay of a Target Region with at Least Two Probes

This example describes exemplary strategies for assaying a target regionusing at least two distinct probes and at least one sink; see FIGS. 13and 14.

FIG. 13 is a schematic flowchart illustrating a strategy for assay of atarget or target region using at least two distinct probes 52 a and 52b. The strategy may be performed in partitions, such as droplets, or maybe performed in a bulk phase.

Probe 52 a may be a primer labeled with a photoluminophore 60 a. Theprobe may be complementary to a sink 54 to form a reporter 130 at thedetection temperature, but not at the annealing temperature used foramplification. Probe 52 a may be configured to bind to a template 80providing a target 82.

Probe 52 b may be a self-quenching probe having a photoluminophore 60 band a quencher 62 b for the photoluminophore. Photoluminophores 60 a and60 b may be optically distinguishable, such as having distinct emissionspectra. A sink capable of binding to probe 52 b may or may not bepresent.

Amplification of target 82 with probe 52 a and reverse primer 90 (R)produces copies of amplicon 140 each labeled with photoluminophore 60 a.Amplification also may produce degraded probe 70 b from probe 52 b, suchthat photoluminophore 60 b is no longer quenched by quencher 62 b. Atthe detection temperature, after amplification, positive partitions maycontain labeled amplicon 140, degraded probe 70 b, and sink 54, andoptionally may contain intact probe 52 b (a remaining amount notdegraded during amplification) and/or reporter 130 (including copies ofprobe 52 a not used for amplification). The positive partitions may havea distinctive luminescence signature produced by light emitted byphotoluminophores 60 a and 60 b. Negative partitions may containsubstantially more reporter 130 and intact probe 52 b than positivepartitions. In some cases, other targets may be assayed in the samepartitions using probes labeled with only photoluminophore 60 a or onlyphotoluminophore 60 b.

FIG. 14 shows another schematic flowchart illustrating another strategyfor assay of a target or target region using at least two distinctprobes 52 a and 52 b. The strategy may be performed in partitions, suchas droplets, or may be performed in a bulk phase.

Each of probes 52 a and 52 b may be a forward primer (F1 and F2) labeledwith a distinct photoluminophore 60 a or 60 b. Each probe 52 a and 52 bmay be at least partially complementary to a respective sink, 54 a and54 b, to form a base-paired form of reporters 130 a, 130 b at thedetection temperature, but not at the annealing temperature used foramplification. Sinks 54 a and 54 b may contain quenchers 62 a and 62 b,which may be copies of each other or structurally different (as shownhere). Each probe 52 a and 52 b may be configured to bind to a template80 providing a target 82, and may be extendable by polymerase. Theprobes may bind to the same strand or respective complementary strandsof the template. In some cases, the probes may function as respectivesense and antisense primers that define the ends of a correspondingamplicon.

Amplification of target 82 with probe 52 a and reverse primer 90 (R)produces copies of amplicon 140 each labeled with photoluminophore 60 a.Amplification also may produce degraded probe 70 b from probe 52 b, suchthat photoluminophore 60 b is no longer quenched by quencher 62 b. Thebase-paired configuration of reporters 130 a and 130 b may be presentafter amplification at a detection temperature, in amounts inverselyrelated to the extent of target amplification.

FIG. 14A shows still another strategy for assay of a target using atleast two distinct probes 52 a and 52 b (of respective reporters 130 a,130 b). The strategy of FIG. 14A is similar to that of FIG. 14 exceptthat probes 52 a, 52 b are configured as a forward primer (F) and areverse primer (R), respectively, instead of as two different forwardprimers. Each probe may be bound by a sink 54 a or 54 b that affectsphotoluminescence of a respective different photoluminophore 60 a or 60b. Amplification of the target may be detectable as a change inphotoluminescence from both reporters 130 a and 130 b (e.g., from bothphotoluminophores 60 a and 60 b). This strategy may be utilized toachieve multiplexing to detect a plurality of different targetsequences, such as in the same set of partitions. As an example, theprimers for five different targets may be labeled as follows with fourdifferent photoluminophores (FAM, HEX, NED, and ALEXA 568), and each maybe base-paired above the detection temperature with a respective sink:

Target No. Label (Forward Primer) Label (Reverse Primer) 1 FAM None 2None HEX 3 FAM HEX 4 FAM NED 5 FAM ALEXA 568Photoluminescence from the various photoluminophores may be detected inone or more wavelength regimes, such as in a pair of detection channelsrepresenting different wavelengths, to allow generation of atwo-dimensional plot of the photoluminescence data. Each of thedifferent combinations of the photoluminophores may produce adistinguishable target-positive cluster for each different target in theplot.

Example 8. Selectively Quenching a Primer Dimer

This example describes an exemplary strategy for a target assayperformed with a primer containing a quencher, to selectively quenchlight emission from a primer dimer 240 relative to a desired amplicon140; see FIG. 15.

The formation of a primer dimer in an amplification assay, generally asan unwanted side-reaction product, can increase the noise and decreasethe accuracy of the assay. FIG. 15 show a strategy for reducing thesignal produced by a primer dimer in partitions (or a bulk volume).Amplification of a target 82 may be performed with a probe 52 serving asa forward primer (F), and a sink 54 b configured to act as a reverseprimer (R) and containing a quencher 62 b for a photoluminophore 60 ofthe probe. Two exemplary products labeled with photoluminophore 60 maybe produced, namely, amplicon 140 and primer dimer 240. Each of theproducts also contains quencher 62 b. However, the respective averagedistance, d1 and d2, between the photoluminophore and quencher may besubstantially greater in amplicon 140 than in primer dimer 240.Accordingly, emission of light from amplicon 140 is quenched much lessthan emission of light from primer dimer 240. As a result,target-negative partitions containing only primer dimer are less likelyto be erroneously assigned as target-positive, which may improve theaccuracy of the assay.

The idea is to intentionally lower the luminescence of primer dimers sothey do not have the ability to affect quantification of the target. Inother words, if a droplet that is somewhat positive due to primer dimerluminescence is classified as a positive droplet, this is not desirableas it affects quantification. The strategy described here can reduce theluminescence of primer dimer containing droplets and allows for moreaccurate target quantification.

The reverse primer has a quencher on it. For normal-sized amplicons, thedistance between the quencher and the photoluminophore on the otherprimer is so great that little or no quenching occurs. In contrast, ifthe primer creates a primer dimer, then the distance is short enoughthat quenching occurs, and the droplets have lower luminescence and arecorrectly classified as negative for the target.

Example 9. Comparison of Background Signals in Singleplex and MultiplexAssays

This example describes exemplary data collected in singleplex andmultiplex assays performed with various combinations of primers andtemplates; see FIGS. 16-18.

Maintaining a good signal-to-noise ratio in multiplex analysis can bechallenging. FIG. 16 shows a pair of plots of fluorescence intensitysignals (fluorescence (fl.) amplitude) detected from droplets travelingthrough a detection region. Each droplet detected may generate acharacteristic fluorescence profile termed an “event,” which is plottedin FIG. 16 (and FIGS. 17 and 19) according to the order in the eventswere detected. In other words, the first event detected is plotted asevent number one, the second event as event number two, and so on, withfluorescence amplitude plotted in FIG. 16 for over 4×10⁵ events(droplets). For each droplet, the fluorescence amplitude measured ineach of two detection channels, channel 1 and channel 2, is plotted.Channel 1 (the “FAM channel”) represent signals detected from one(1-plex) or five probes (five-plex) containing a FAM dye, and channel 2(“the HEX channel”) represents signals detected from a single probecontaining a HEX dye in a single target assay. The data collected in theHEX channel serves as a control.

The data shown in the top plot was obtained with singleplex assays (eachassay is designed to amplify and detect only one target) and multiplexassays (each assay is a combination of assays designed to amplify anddetect multiple targets. The two types of assays are identified abovethe top plot in the row labeled “N-plex,” with “1” indicating asingleplex assay and “5” indicating a multiplex assay for five targets.Each singleplex target assay and corresponding template has beenassigned a number for one to five. The particular single template addedto each assay, to generate amplification-positive droplets, isidentified in the row labeled “template.” Accordingly, each of templates“1” through “5” was added individually to a singleplex assay for thecorresponding target, and was the only template added to a five-plexassay for all of targets 1 to 5. In other words, amplification of eachof templates 1 to 5 was compared in a singleplex assay and a five-plexassay, to test the effect of multiplexing on the background signal andsignal-to-noise ratio. The band of droplets negative for the particulartemplate being tested is identified immediately to the right of eachnegative droplet band with an empty rectangle, and the droplets positivefor the template with a filled rectangle.

Each of the singleplex assays exhibit good signal-to-noise ratios, withthe band of positive droplets (filled rectangle) well resolved from theband of negative droplets (empty rectangle). However, when the sametarget is tested in a five-plex assay with only the correspondingtemplate present, the signal-to-noise ratio becomes much less favorable.When tested in a multiplex assay, it is difficult to separate thepositive droplets (signal) from the negative droplets (noise) in threeof the five multiplex assays (templates 1, 2, and 4), making accuratequantification difficult or impossible. Multiplexing may cause highbackground because components of different assays can interact with eachother to raise the noise beyond what would be expected from standardadditive noise from each assay.

FIG. 17 shows data collected from droplets, generally as in FIG. 16,from a set of five singleplex assays each for a different target, afive-plex assay for five targets, and various 4-plex assays for fourtargets. Template is present in only one of the conditions for thevarious assays represented in the top plot (“P+T+”), and template ispresent in a subset of conditions in the bottom plot (i.e., the HEXchannel control). Positive and negative bands of droplets are identifiedwith filled and empty rectangles as in FIG. 16.

Each of the five singleplex assays (1 to 5 for different targets)exhibits a low background signal. In contrast, the background levelincreases dramatically when the five assays are combined as a five-plexassay. The high five-plex background is observed without polymerase ortemplate (“P−T−”), with polymerase and without template (“P+T−”), andwith polymerase and template (“P+T+”). Removing the primers and probefor each of the five assays individually from the five-plex assay tocreate five different four-plex assays, each missing one of the assays,shows that each of the assays contributes to the high background level.

The additive noise of the 5 singleplex assays is 2598.6, whereas theobserved noise of the five-plex assay is 6863.0. Combining these assayscreates an additional 4264.4 of noise, which more than doubles the noisein the system over additive noise.

FIG. 18 shows possible configurations of probe 52 in an ideal singleplexassay and a non-ideal singleplex or multiplex assay. The probe may havea smaller average spacing between photoluminophore 60 and quencher 62 inthe ideal singleplex assay relative to the non-ideal singleplex ormultiplex assay. For example, a primer 260 in assay may have a tendencyto bind the probe, which may increase the rigidity of the probe, to keepphotoluminophore 60 and quencher 62 farther apart. The greater thenumber of targets being quantified, the greater the chance that a probefrom one assay will be bound by a component of another assay. Detectionmay be performed at a temperature much lower than the temperatures usedfor amplification, such as at room temperature, thereby allowing amismatched reporter 262 to form. Accordingly, an exemplary theory behindthe greater than additive noise observed is that components from thedifferent assays interact with each other to increase probe rigidity,which reduces quenching and drives up the noise.

Example 10. Test of a Sink that Promotes Intermolecular Quenching

This example describes exemplary data collected from droplets formed tocontain a reporter according to Example 5 (FIG. 11); see FIG. 19.

The plot of FIG. 19 shows data collected from droplets each containingeither a five-plex assay as in FIGS. 16 and 17 or a singleplex assay. Notemplate was added, so each band on the plot represents negativedroplets, as indicated by an unfilled rectangle to the right of eachband. In the multiplex assay, each of the assays targets a differentgene. The primer and probe concentrations for each assay were 900 nM and250 nM, respectively. A sink was included in the droplets at aconcentration of 500 nM, where indicated. The sink was designed as inFIG. 11 and binds the probe for only one target assay. Use of the sinkdecreases the fluorescence of the negative droplets in both thesingleplex assay and the multiplex assay (even though the sink binds toonly one of the five probes used in the multiplex assay). Accordingly,the sink can reduce the amplitude of negative droplets (noise) inmultiplex and singleplex droplet PCR analysis. Addition of sinks thatbind to others of the five probes can reduce background further.

Example 11. Comparison of Different Probes in Droplet-Based Assays

This example compares distinct strategies for assay of a target regionin droplets; see FIGS. 20-22.

FIG. 20 shows a schematic flowchart illustrating a pair of distinctstrategies for assay of respective, overlapping target regions 82 a and82 b in partitions (e.g., droplets).

The strategy on the left, strategy 280, involves a labeled primer (probe52 a) and a sink 54. Strategy 280 can quantify target region 82 a oftemplate 80 and utilizes probe 52 a acting as a forward primer togenerate labeled amplicon 140 a that is not quenched. The unused probethat is not incorporated can be bound by sink 54 to form quenchedreporter 130 at the detection temperature.

The strategy on the right, strategy 290, involves a self-quenched probe52 b that is not an amplification primer. The probe may, for example, bea Taqman® probe. Strategy 290 can quantify target region 82 b oftemplate 80 and utilizes probe 52 b that binds intermediate a pair ofamplification primers (F and R). The amplification primers define anamplicon that encompasses the amplicon of strategy 280. Probe 52 b iscleaved during amplification to form a degraded probe 70 b that is notquenched by quencher 62. Residual, unused probe also may be present,particularly in target-negative partitions.

Fragmentation of DNA can artificially reduce concentration measurementswhen using PCR-based methods. Many sources of DNA such as FFPE(formalin-fixed paraffin-embedded) or environmental samples are highlyfragmented. One possible approach to alleviate the errors caused byfragmentation is to create assays that are as short as possible.Strategy 280 has advantages over strategy 290 in this regard. Forexample, use of a probe that serves as an amplification primer allowsthe forward and reverse primers to be positioned more closely to oneanother than when the probe binds intermediate the pair of amplificationprimers. Accordingly, a shorter target region (e.g., less than about 75,60, or 50 nucleotides) can be quantified with the strategy on the left,which reduces the number of target copies that are missed due to randombreakage during isolation/preparation of nucleic acid that provides thetarget.

FIG. 21 shows a plot of fluorescence data collected from sets ofdroplets assayed according to strategy 280 (“labeled primer+sink”) orstrategy 290 (“self-quenched probe”) of FIG. 20, with primers for aCCND1 target. The oligonucleotides used for strategy 280 are as follows:5′-FAM-TATCTGAGGGGCGGGAGAG-3′, probe/forward primer (SEQ ID NO:1);5′-GAGGTCACGACATTTTAGCG-3′, reverse primer (SEQ ID NO:2); and5′-CGCCCCTCAGATA-IB-3′, sink, where IB is Iowa Black® dark quencher (SEQID NO:3). The primers used for strategy 290 are as follows:5′-ACATTGATTCAGCCTGTTTGG-3′, forward primer (SEQ ID NO:4);5′-GAATTCATCGGAACCGAACTT-3′, reverse primer (SEQ ID NO:5); and5′-FAM-TCCTTGCACCCATGCCTGTCCA-IB-3′, probe (SEQ ID NO:6).

Eight sets of droplets (identified with the numbers 1-8) were processedaccording to each strategy, with amplification promoted by thermocyclingat a distinct annealing temperature for each set of droplets. Moreparticularly, set 1 was an annealed at 65° C., set 8 at 55° C., and sets2-7 at temperature increments between the annealing temperatures of sets1 and 8. The droplets in each set were passed through a detectorserially and were detected as individual “events” based on afluorescence amplitude measured from each droplet. The detected dropletsor “events” are numbered in sequence on the graph and are plotted asindividual points. Target-negative droplets of the set produce a lowerfluorescence amplitude, identified by an unfilled rectangle, andtarget-positive droplets of the set produce a higher fluorescenceamplitude, identified by a filled rectangle. The presence of dropletshaving an intermediate fluorescence amplitude (“rain”) is reducedsubstantially by decreasing the annealing temperature, as seen in sets5-8 for each strategy.

FIG. 22 shows a graph of target concentrations determined from the dataof FIG. 21, for each set of droplets. Sets 5-8 for each assay strategygenerated substantially the same target concentration. The experimentspresented in this example show that an AMP assay with a sink produced anequivalent concentration measurement to a TaqMan® assay for the samegene, CCND1. The AMP assay does not require space for an additionalprobe and can amplify a significantly shorter product than a TaqMan®assay. Furthermore, AMP assays take advantage of contact quenching andare easily amenable to multiplexing.

Example 12. Test of a Sink that Binds a Pair of Self-Quenched Probes

This example describes exemplary data collected from droplets formed tocontain self-quenched probes 52 a and 52 b that are specific,respectively, for a wild type target 82 a and a mutant target 82 b. Eachof probes 52 a and 52 b may be bound by the same sink 54 at a detectiontemperature to form respective quenched reporters 130 a and 130 b. Theassay strategy is generally as described in Section I (e.g., see FIGS.1-3) and elsewhere in the present disclosure; see FIGS. 23 and 24.

FIG. 23 shows a schematic of a strategy for performing a multiplex assay(A146T assay) of wild-type and mutant alleles of a K-Ras gene indroplets. Probes 52 a and 52 b and primers (F and R) are aligned withtheir prospective binding sites in wild-type and mutant templates 80 aand 80 b. Targets 82 a and 82 b may differ at a single nucleotide (ormore than one nucleotide), identified at 300 in the mutant template.

Droplets were formed with one pair of primers for amplification of thesame region of K-Ras from wild-type and mutant templates. The sequencesof the primers are as follows: 5′-AGAAGCAATGCCCTCTCAAG-3′, forwardprimer (SEQ ID NO:7); and 5′-AAACAGGCTCAGGACTTAGC-3′, reverse primer(SEQ ID NO:8). The assay used a wild-type probe 52 a and a mutant probe52 b labeled with distinct fluorophores, namely, HEX dye (60 a) and FAMdye (60 b), respectively, and each conjugated to the same quencher, IOWABLACK® dark quencher (“IB”). The respective sequences of probes 52 a and52 b are as follows: 5′-HEX-ATTGAAACATCAGCAAAGACAAGACA-IB-3′ (SEQ IDNO:9); and 5′-FAM-TTGAAACATCAACAAAGACAAGACAGG-IB-3′ (SEQ ID NO:10). Theprobes have a single nucleotide difference that allows the probes toselectively bind to wild-type or mutant template/amplicon. Differentsets of droplets were formed to contain either no sink 54 or the sink attwo different ratios of probe to sink (1:1 and 1:2). The sink isperfectly complementary to the mutant probe (no mismatches). However,the sink binds substantially to both probes at the detection temperatureused (room temperature), and thus is not selective for the mutant probe.The sequence of the sink is 5′-GTTGATGTTTCAA-IB-3′ (SEQ ID NO:11).

FIGS. 24A-C show scatter plots of fluorescence data collected fromdroplets formed without a sink (A) and with different amounts of thesink (B and C). Each droplet produces a point in the plot, with theposition of the point determined by the channel 1 fluorescence amplitude(HEX dye, wild-type probe) of the droplet on the X-axis and the channel2 fluorescence amplitude (FAM dye, mutant probe) of the same droplet onthe Y-axis. The droplets form four clusters or populations, which arecolor-coded and located in four quadrants: black (−/−) for doublenegative (neither target present), blue (+/−) for single positive withonly the mutant target present, green (−/+) for single positive withonly the wild-type target present, and brown (+/+) for double positive(mutant and wild-type targets present). In FIG. 24A, without a sink, thebasal fluorescence of negative droplets is relatively high—thefluorescence amplitude difference between negative and positive dropletsfor each target is only about two-fold. In FIGS. 24B and 24C, the sinkproduces a substantial decrease in the background fluorescence ofdroplets negative for either or both targets. The sink produces arelatively insubstantial reduction in the fluorescence amplitude ofdroplets positive for either or both targets.

Example 13. Test of a Sink for a Probe Containing No Quencher

This example describes exemplary data collected from droplets formed tocontain a probe and a sink and then processed to promote amplification,generally according to Example 2 (e.g., see FIG. 8); see FIGS. 25 and26.

FIG. 25 shows a schematic of another strategy for performing a multiplexassay (A146T assay) of wild-type and mutant alleles of a K-Ras gene indroplets. The strategy is substantially the same as in FIG. 23, exceptthat each probe 52 a and 52 b is labeled with a photoluminophore but nota quencher. The sequences of probes 52 a and 52 b are5′-HEX-ATTGAAACATCAGCAAAGACAAGACA-3′ (SEQ ID NO:12) and5′-FAM-TTGAAACATCAACAAAGACAAGACAGG-3′ (SEQ ID NO:13). The sink (SEQ IDNO:11) of the preceding example was used here, too.

FIG. 26 present fluorescence data collected from droplets formedaccording to the strategy of FIG. 25. The data is plotted as describedabove for FIG. 24 and shows that intermolecular quenching of probes witha sink, without probe self-quenching, resolves target populations intodistinct clusters.

Example 14. Allele-Specific Amplification

This example describes an exemplary strategy for allele-specificamplification in the presence of a sink that binds allele-specificprobes that function as primers; see FIG. 27.

FIG. 27 shows a schematic of a partition-based strategy for performing aduplex assay of wild-type and mutant alleles of a gene. The alleles maydiffer by any suitable number of nucleotides, such as a singlenucleotide polymorphism 300, as shown here. A wild-type target 82 a maybe amplified from a wild-type template 80 a with a first pair ofprimers, namely, a forward primer (Fa) (probe 52 a) and a reverse primer(Ra), that each prime specifically on the wild-type template relative tothe mutant template. A mutant target 82 b may be amplified from a mutanttemplate 80 b with a different pair of primers, namely, a forward primer(Fb) (probe 52 b) and a reverse primer (Rb), that each primespecifically on the mutant template relative to the wild-type template.Each forward primer and each reverse primer may end on the variantnucleotide position as shown, such that the primers overlap by at leastone nucleotide. Probes 52 a and 52 b may be labeled with respectivephotoluminophores 60 a and 60 b that are optically distinguishable. Theprobes may be quenched intermolecularly at room temperature, asreporters 130 a and 130 b, by binding to copies of the same sink 54,which is complementary to each probe. In other embodiments, eitherallele may be quantified in a singleplex assay.

In other embodiments, unlabeled primers may be used for allele-specificamplification, and a generic reporter (e.g., an intercalating dye) maybe used to label one or more amplicons produced by amplification of oneor both targets. Both targets may be assayed together in a multiplexassay by adjusting one or more primer concentrations and/or primermelting temperatures to render the signal amplitude of partitionspositive for one of the targets distinguishable from the signalamplitude of partitions positive for the other target (and, optionally,distinguishable from the signal amplitude of partitions positive forboth targets).

Example 15. Probe Displacement Assays without Probe Degradation

This example describes an exemplary strategy for a target assay in whichprobe is displaced but not degraded as primer is incorporated intoamplicon; see FIGS. 28-31.

FIG. 28 shows exemplary reporters 350 a,b used in the assay. Thereporters each include a probe 352 a,b and a primer 354 a,b. The probes,in turn, each include at least a probe oligomer 358 a,b, one or moreluminophores 360 a,b, and one or more quenchers 362 a,b. The primers, inturn, each include at least a primer oligomer 364 a,b. The probeoligomer and the primer oligomer for each reporter may be partially orfully complementary and are capable of base-pairing with one anotherbelow a melting temperature of the reporter. The luminophore(s) andquencher(s) are positioned on each probe such that the probe is moreluminescent (i.e., produces more photoluminescence) when the probe isbound to the respective primer than when the probe is unbound.Specifically, when the probe is bound to the primer, the luminophore(s)and quencher(s) are held apart, reducing the effect of the quencher onluminescence of the luminophore. In contrast, when the probe is unbound,either because the system is above the melting point of the probe andprimer pair, or because at least some primer is unavailable to bindprobe because the primer has been incorporated into amplicon duringamplification, the luminophore and quencher are not held apart (althoughstill bound to the same oligonucleotide) and may come into sufficientproximity that the quencher reduces or eliminates luminescence from theluminophore. Luminophore and quencher may be positioned, in any suitablenumber each, at any suitable positions on the probe capable of yieldingthe effect described above.

FIGS. 28 and 29 show exemplary configurations produced by amplification.Fluorescence typically is detected at the completion of theamplification reaction and, for droplet-based assays, in a sample thathas been partitioned into droplets before amplification. FIG. 28 showsconfigurations produced in the absence of target, such as target DNA ortarget RNA. Here, the probe remains duplexed with the primer such thatthe associated luminophore and quencher are fixed at a distance from oneanother, leading to higher fluorescence. In a droplet assay, theseconfigurations would correspond to no-target droplets or no DNA/no RNAcontrol droplets. FIG. 29, in contrast, shows configurations produced inthe presence of target. Here, primer has been incorporated intoamplicons, leaving excess probe free-floating and self-quenching. Inother words, in samples, such as droplets, that have target DNA ortarget RNA, probe will be displaced from primer as primer becomes a partof the (PCR−) amplified product. Once the probe is displaced from theprimer, the quencher and luminophore will be free to interact directlywith one another, allowing the quencher to quench the fluorescence ofthe luminophore and reduce fluorescence. Thus, positive signal (i.e.,signal from samples positive for target, in which amplificationoccurred) will be lower than negative signal (i.e., signal from samplesnegative for target, in which amplification did not occur). Total assaysignal, including both positive signal and negative signal, can beincreased by using the same fluorophore for both primer-probe pairs(e.g., for forward and reverse primers). Conversely, total assay signalcan be decreased by omitting fluorophore (or both fluorophore andquencher) from one of the primer-probe pairs.

FIG. 30 shows representative data, plotted in two dimensions, from anassay such as the assay of FIGS. 28 and 29 obtained using a FAM-labeledprobe partially complementary to one primer and a HEX-labeled probepartially complementary to the other primer. Panel A shows results froma sample containing target DNA. The results include data clusters frompartitions both negative for (“negative cluster”) and positive for(“positive cluster”) the target DNA. The fluorescence of the negativecluster (in which probe and primer remain bound) is higher than thefluorescence of the positive cluster (in which probe is unbound or freein solution). Panel B shows results from a control well not containingDNA. The negative sample or no-target sample has only the higherfluorescent cluster of droplets.

FIG. 31 illustrates how displacement probe assays may be used incombination with traditional TaqMan® assays to create a space for thepositives 360 degrees around the central negative cluster. The signalshift may be achieved through mixing different fluorophores or dyes oneach probe as well as assay types. In other words, signal may bepurposely altered by adding a weak or strong quencher and/or mixed byusing one fluorophore for one primer-probe pair and a differentfluorophore for the other primer-probe-pair. This, in turn, shouldincrease multiplexing.

The assays described in this example may have several advantages. First,because probe and primer bind to one another, allowing probe and primerto utilize the same sequence space, the probes may be used for thedetection of nucleic acids that are too short for a traditional TaqMan®assay (FIG. 28). Second, the method provides the potential to multiplexby allowing for the positive droplets to occupy a space in 360 degreesaround the negative signal (FIG. 31). These probe displacement assaysmay be used for any suitable application; however, they are especiallyuseful for (1) detection of targets that are only long enough for aprimer to be used on each side which is approximately 15-40 nucleotideslong, (2) increasing the number of amplifiable copies of DNA or RNA in afragmented or damaged sample, and (3) micro RNAs that are 15-25nucleotides long.

The assays in this example (and other examples) may be modified byreplacing fluorescence quencher with a fluorescence enhancer, such as anantenna that transfers energy to, rather than siphoning energy from, thefluorophore. (This may be accomplished, for example, by using a donorand acceptor pair, then exciting both donor and acceptor, and finallydetecting acceptor fluorescence.) This would have the effect, in thisexample, of reversing the relative intensities of the positive andnegative signals, because amplification leading to unbound probe wouldbring fluorophore and enhancer in proximity, increasing rather thandecreasing fluorescence.

Example 16. Selected Embodiments I

This example describes selected embodiments of assay methods andcompositions, presented as a series of indexed paragraphs.

1. A method of performing an assay, the method comprising: (A) providingpartitions including a target at partial occupancy, a probe including aphotoluminophore, and a sink that binds the probe to reduce an abilityof the photoluminophore to emit light; (B) amplifying the target in thepartitions; (C) detecting light that is dependent at least in part on apresence of the photoluminophore in the partitions; and (D) determininga level of the target present in the partitions based on the lightdetected.

2. The method of paragraph 1, wherein the step of amplifying isperformed with one or more primers, and wherein the probe acts as one ofthe one or more primers for the step of amplifying.

3. The method of paragraph 2, wherein the step of amplifying isperformed with a first primer and a second primer that define oppositeends of an amplicon produced by the step of amplifying, wherein theprobe is the first primer, and wherein the second primer is conjugatedto a quencher for the photoluminophore.

4. The method of paragraph 3, wherein the sink includes anoligonucleotide that is complementary to the first primer.

5. The method of paragraph 4, wherein the oligonucleotide is conjugatedto a quencher for the photoluminophore.

6. The method of any of paragraphs 1 to 5, wherein the step of providingpartitions includes a step of providing partitions containing a pair ofprobes, wherein the step of amplifying is performed with the pair ofprobes each including a distinct photoluminophore and acting as a primerfor a distinct target, and wherein the step of determining includes astep of determining a level of each of the distinct targets.

7. The method of paragraph 1, wherein the probe is a first probeincluding a first photoluminophore, further comprising a second probeincluding a distinct second photoluminophore and configured to bind tothe target at a different position than the first probe.

8. The method of paragraph 7, wherein the second probe is not bound bythe sink.

9. The method of paragraph 8, wherein the second probe is conjugated toa quencher.

10. The method of any of paragraphs 7 to 9, wherein the second probe isa primer that is extended during the step of amplifying.

11. The method of paragraph 10, wherein each of the first probe and thesecond probe is a distinct primer that is extended during the step ofamplifying.

12. The method of any of paragraphs 7 to 11, wherein the step ofdetermining a level of the target is based on light detected from thefirst photoluminophore of the first probe and the secondphotoluminophore of the second probe.

13. The method of paragraph 7, wherein only one of the first probe andthe second probe is a primer that is extended during the step ofamplifying.

14. The method of paragraph 13, wherein neither the first probe nor thesecond probe is a primer that is extended during the step of amplifying.

15. The method of any of paragraphs 1 to 14, wherein the partitions aredroplets.

16. The method of any of paragraphs 1 or 15, wherein the probe includesa probe oligonucleotide, wherein the sink includes a sinkoligonucleotide, and wherein the probe oligonucleotide is longer thanthe sink oligonucleotide and/or is configured to form a greater numberof base pairs with the target than with the sink oligonucleotide.

17. The method of any of paragraphs 1 to 16, wherein a plurality of thepartitions contain no copies of a template corresponding to the target.

18. The method of any of paragraphs 1 to 17, wherein a plurality of thepartitions contain only one copy of a double-stranded or single-strandedtemplate corresponding to the target.

19. The method of any of paragraphs 1 to 18, wherein the step ofproviding partitions includes a step of providing partitions in whichthe target is formed by a pair of complementary strands.

20. The method of any of paragraphs 1 to 19, wherein the sink includesan oligonucleotide, and wherein the oligonucleotide binds specificallyto the probe.

21. The method of any of paragraphs 1 to 20, wherein the sink includes aquencher for the photoluminophore.

22. The method of paragraph 21 wherein the quencher is not substantiallyphotoluminescent.

23. The method of paragraph 21, wherein the quencher isphotoluminescent, and wherein the step of detecting light includes astep of detecting light emitted by the quencher.

24. The method of any of paragraphs 20 to 23, wherein the quencher isconjugated to a 3′-end of the oligonucleotide.

25. The method of any of paragraphs 20 to 23, wherein the quencher is atan internal position between the 5′-end and the 3′-end of theoligonucleotide.

26. The method of any of paragraphs 1 to 25, wherein at least twodistinct targets are amplified in the partitions, wherein one or more ofthe partitions contain each of the targets, and wherein a level of eachtarget is determined.

27. The method of any of paragraphs 1 to 26, wherein the sink does notinclude a quencher for the photoluminophore.

28. The method of any of paragraphs 1 to 27 wherein the photoluminophoreis a fluorophore, and wherein the step of detecting light includes astep of detecting fluorescence of the fluorophore.

29. The method of any of paragraphs 1 to 28, wherein the probe iscapable of specifically binding a region of the target.

30. The method of any of paragraphs 1 to 29, wherein the probe includesan oligonucleotide, and wherein the quencher and the photoluminophoreare connected to respective opposite ends of the oligonucleotide.

31. The method of any of paragraphs 1 to 30, wherein the step ofproviding partitions includes a step of providing partitions surroundedby a continuous liquid phase.

32. The method of paragraph 31, wherein the continuous liquid phase isimmiscible with the partitions.

33. The method of paragraph 31 or 32, wherein the continuous liquidphase is composed at least predominantly of oil.

34. The method of any of paragraphs 1 to 33, wherein the step ofproviding partitions includes a step of providing partitions in whichthe sink is present at a higher concentration than the probe.

35. The method of any of paragraphs 1 to 34, wherein the sink isconfigured to promote intramolecular quenching of the probe.

36. The method of paragraph 35, wherein a single copy of the sink isconfigured to circularize a single copy of the probe.

37. The method of paragraph 35, wherein a single copy of the sink isconfigured to bind two copies of the probe, and wherein a single copy ofthe probe is configured to bind two copies of the sink.

38. The method of any of paragraphs 1 to 37, wherein the step ofproviding partitions includes a step of providing partitions containingmore copies of the sink than the probe.

39. The method of any of paragraphs 1 to 38, wherein the step ofproviding partitions includes a step of providing partitions containingan amount of the sink sufficient to bind substantially all of the probein each partition.

40. The method of any of paragraphs 1 to 39, wherein the step ofamplifying includes a step of thermally cycling the partitions.

41. The method of paragraph 40, wherein the step of amplifying includesa step of performing a polymerase chain reaction.

42. The method of any of paragraphs 1 to 41, wherein the step ofamplifying includes a step of generating an amplicon corresponding tothe target, and wherein the probe binds to a region of the amplicon.

43. The method of any of paragraphs 1 to 42, wherein the step ofdetecting light is performed after amplification of the target hasreached an endpoint.

44. The method of any of paragraphs 1 to 43, wherein the light detectedis emitted at least in part by the photoluminophore.

45. The method of any of paragraphs 1 to 43, wherein the light detectedis emitted at least in part by an energy transfer partner of thephotoluminophore

46. The method of any of paragraphs 1 to 45, further comprising a stepof irradiating the partitions with light that excites thephotoluminophore

47. The method of any of paragraphs 1 to 46, further comprising a stepof determining a fraction of partitions that are positive or that arenegative for amplification of the target, and wherein the step ofdetermining a level is based on the fraction.

48. The method of any of paragraphs 1 to 47, wherein the step ofdetermining a level includes a step of determining a concentration ofthe target.

49. The method of any of paragraphs 1 to 48, wherein the sink binds tothe probe to form a reporter having a melting temperature, and whereinthe step of amplifying is performed with the partitions maintained abovethe melting temperature of the reporter.

50. The method of paragraph 49, wherein the melting temperature is lessthan 50 degrees Celsius.

51. The method of paragraph 49 or 50, wherein the step of detectinglight is performed with the partitions below the melting temperature ofthe reporter.

52. The method of paragraph 1, wherein the partitions contain templatecopies for a first allele and/or a second allele of the target, whereinthe step of amplifying is performed with a forward primer and a reverseprimer that are each selectively extendable when bound to template forthe first allele relative to template for the second allele, and whereinthe probe is the forward primer or the reverse primer.

53. The method of paragraph 52, wherein the forward primer and thereverse primer overlap by at least one nucleotide.

54. The method of paragraph 52, wherein the forward primer and thereverse primer overlap at a site of nucleotide variation between thefirst and second alleles.

55. The method of paragraph 54, wherein each of the forward primer andthe reverse primer ends at the site of nucleotide variation.

56. The method of any of paragraphs 52 to 55, wherein the sink includesa quencher for the photoluminophore.

57. The method of any of paragraphs 52 to 56, wherein the probe is afirst probe, further comprising a second probe that is extendableselectively as a primer when bound to template for the second allelerelative to template for the first allele.

58. The method of paragraph 57, wherein the sink binds to the secondprobe.

59. A method of performing an assay, the method comprising: (A)providing droplets including a target at partial occupancy, a probehaving a photoluminophore and a quencher for the photoluminophore, andan oligonucleotide that binds the probe to reduce an ability of thephotoluminophore to emit light; (B) amplifying the target in thedroplets; (C) detecting light emitted at least in part by thephotoluminophore; and (D) determining a level of the target based on thelight detected.

60. A method of performing an assay, the method comprising: (A) forminga reaction mixture including a target, a probe having a photoluminophoreand a quencher for the photoluminophore, and a first oligonucleotidethat binds the probe to reduce an ability of the photoluminophore toemit light, wherein the probe includes a second oligonucleotideconfigured to bind a region of an amplicon; (B) amplifying the target togenerate the amplicon; and (C) detecting light emitted at least in partby the photoluminophore.

61. The method of paragraph 60, further comprising a step of formingpartitions containing portions of the reaction mixture, wherein the stepof amplifying is performed in a plurality of the partitions, and whereinthe step of detecting light includes a step of detecting light from aplurality of the partitions.

62. The method of paragraph 60 or 61, wherein the step of formingpartitions includes a step of fusing droplets.

63. The method of any of paragraphs 60 to 62, wherein the step offorming partitions includes a step of forming droplets.

64. The method of any of paragraphs 60 to 63, wherein the reactionmixture is a dispersed phase of an emulsion.

65. A method of performing an assay, the method comprising: (A) forminga reaction mixture including a template for a target, a probe includinga photoluminophore, and a sink that binds the probe to reduce an abilityof the photoluminophore to emit light; (B) amplifying the target in thereaction mixture, at least in part by extending the probe; (C) detectinglight that is dependent at least in part on a presence of thephotoluminophore in the reaction mixture; and (D) determining a level ofthe target based on the light detected.

66. The method of paragraph 65, wherein the step of amplifying generatesan amplicon and is performed with a first primer and a second primerthat define respective ends of the amplicon, wherein the probe is thefirst primer, and wherein the second primer is conjugated to a quencherfor the photoluminophore.

67. The method of paragraph 65 or 66, wherein the sink includes aquencher for the photoluminophore.

68. A composition, comprising: (A) a template; (B) a probe having aphotoluminophore; and (C) a sink that is complementary to and binds theprobe to reduce an ability of the photoluminophore to emit light.

69. The composition of paragraph 68, further comprising one or moreprimers to amplify a target from the template.

70. The composition of paragraph 68, wherein the probe is a primer foramplification of a target.

71. The composition of any of paragraphs 68 to 70, further comprising apolymerase.

72. The composition of paragraph 71, wherein the polymerase is heatstable.

73. The composition of any of paragraphs 68 to 72, further comprising aligase.

74. The composition of any of paragraphs 68 to 73, further comprisingdNTPs, NTPs, or both.

75. The composition of any of paragraphs 68 to 74, wherein the probeincludes a quencher for the photoluminophore.

76. The composition of paragraph 75, wherein the quencher is a firstquencher, and wherein the sink includes a second quencher for thephotoluminophore.

77. The composition of any of paragraphs 68 to 76, wherein the template,the probe, and the sink are disposed in partitions, further comprising asame liquid continuous phase surrounding each of the partitions.

Example 17. Selected Embodiments II

This example describes selected embodiments of assay methods andcompositions, presented as a series of indexed paragraphs.

1. A method of analysis, the method comprising: (A) forming at least onevolume containing a reporter including a first oligomer and a secondoligomer capable of base-pairing with one another below a meltingtemperature of the reporter to affect a photoluminescence detectablefrom the reporter; (B) amplifying a target in the at least one volume,at least in part by extending one or more primers at a temperature abovethe melting temperature of the reporter; (C) detecting thephotoluminescence of the reporter from the at least one volume,optionally while the at least one volume is at a temperature below themelting temperature of the reporter; and (D) determining a property ofthe target based on the photoluminescence detected.

2. The method of paragraph 1, wherein the step of amplifying uses thefirst oligomer as a primer.

3. The method of paragraph 2 or paragraph 3, wherein the first oligomerincludes a photoluminophore from which the photoluminescence isdetected.

4. The method of any of paragraphs 1 to 3, wherein the first oligomer isfully complementary to the target.

5. The method of any of paragraphs 1 to 4, wherein the second oligomerincludes a photoluminophore from which the photoluminescence isdetected, and wherein the first oligomer includes an energy transferpartner of the photoluminophore.

6. The method of paragraph 5, wherein the energy transfer partner is aquencher.

7. The method of any of paragraphs 1 to 6, wherein the first oligomerhas a longer chain of base-containing units than the second oligomer.

8. The method of any of paragraphs 1 to 7, wherein the first oligomerforms a hybrid with the target having a higher melting temperature thanthe melting temperature of the reporter.

9. The method of paragraph 8, wherein the higher melting temperature isat least ten degrees higher than the melting temperature of thereporter.

10. The method of any of paragraphs 1 to 4 and 7 to 9, wherein the firstoligomer includes a photoluminophore from which the photoluminescence isdetected and also includes an energy transfer partner of thephotoluminophore, and wherein the second oligomer does not include anenergy transfer partner of the photoluminophore.

11. The method of any of paragraphs 1 to 10, wherein thephotoluminescence of the reporter decreases when the first oligomer andthe second oligomer base-pair with one another.

12. The method of any of paragraphs 1 to 4 and 7 to 11, wherein thefirst oligomer includes a photoluminophore from which thephotoluminescence is detected, and wherein an energy transfer partner ofthe photoluminophore is included in the second oligomer.

13. The method of paragraph 12, wherein the energy transfer partner is aquencher.

14. The method of paragraph 12 or paragraph 13, wherein an energytransfer partner of the photoluminophore also is included in the firstoligomer.

15. The method of any of paragraphs 1 to 14, wherein the property is alevel of the target.

16. The method of any of paragraphs 1 to 15, wherein the step ofamplifying causes cleavage of the first oligomer, the second oligomer,or both the first oligomer and the second oligomer, and wherein thecleavage affects the photoluminescence detected.

17. The method of any of paragraphs 1 to 16, wherein the step of formingat least one volume includes a step of forming a plurality of partitionsthat contain the target at partial occupancy.

18. The method of paragraph 17, wherein the step of amplifying includesa step of exposing the plurality of partitions to a plurality of thermalcycles, and wherein the partitions are maintained continuously above themelting temperature of the reporter throughout the plurality of thermalcycles.

19. The method of any of paragraphs 1 to 3 and 5 to 18, wherein thefirst oligomer has (a) a region dedicated to base-pairing with thetarget such that the first oligomer functions as a primer included inthe one or more primers and (b) another region dedicated to base-pairingwith the second oligomer and not the target.

20. The method of any of paragraphs 1 to 19, wherein the first oligomeror the second oligomer includes a chain of base-containing units, andwherein a photoluminophore is attached to a 5′-end of the chain.

21. The method of any of paragraphs 1 to 4 and 7 to 20, wherein thefirst oligomer includes a photoluminophore from which thephotoluminescence is detected, and wherein the second oligomer is fullycomplementary to the target.

22. The method of any of paragraphs 1 to 21, wherein the meltingtemperature of the reporter is less than about 45 degrees Celsius.

23. The method of any of paragraphs 1 to 4 and 6 to 22, wherein thefirst oligomer includes a photoluminophore, wherein the second oligomerincludes a chain of base-containing units, and wherein the secondoligomer includes an energy transfer partner of the photoluminophore.

24. The method of paragraph 23, wherein the energy transfer partner isattached to a 3′-end of the chain.

25. The method of paragraph 23, wherein the energy transfer partner isattached to a nonterminal unit of the chain.

26. The method of paragraph 23, wherein the energy transfer partner isattached to a 5′-end of the chain.

27. The method of any of paragraphs 1 to 26, wherein the second oligomerhas one or more mismatches with the first oligomer when the first andsecond oligomers base-pair with one another.

28. The method of any of paragraphs 1 to 27, wherein each of the firstoligomer and the second oligomer is at least partially complementary tothe target.

29. The method of paragraph 28, wherein the second oligomer has one ormore mismatches with the target.

30. The method of any of paragraphs 1 to 29, wherein a 3′-end of thefirst oligomer is aligned with a 5′-end of the second oligomer when theoligomers base-pair with one another.

31. The method of any of paragraphs 1 to 30, wherein a 5′-end of thefirst oligomer is aligned with a 3′-end of the second oligomer when theoligomers base-pair with one another.

32. The method of any of paragraphs 1 to 29 and 31, wherein a 3′-end ofthe first oligomer is aligned with an interior base-containing unit ofthe second oligomer when the oligomers base-pair with one another.

33. The method of any of paragraphs 1 to 30 and 32, wherein a 5′-end ofthe first oligomer is aligned with an interior base-containing unit ofthe second oligomer when the oligomers base-pair with one another.

34. The method of any of paragraphs 1 to 30, 32, and 33, wherein a3′-end of the second oligomer is aligned with an interiorbase-containing unit of the first oligomer when the oligomers base-pairwith one another.

35. The method of any of paragraphs 1 to 29 and 31 to 34, wherein a5′-end of the second oligomer is aligned with an interiorbase-containing unit of the first oligomer when the oligomers base-pairwith one another.

36. The method of any of paragraphs 1 to 35, the reporter being a firstreporter, further comprising a second reporter including a pair ofoligomers capable of base-pairing with one another below a meltingtemperature of the second reporter to affect a photoluminescencedetectable from the second reporter and optionally distinguishable fromthe photoluminescence detectable from the first reporter, wherein thestep of amplifying a target includes a step of extending a forwardprimer provided at least in part by one of the reporters and a step ofextending a reverse primer provided at least in part by the otherreporter, and wherein the property of the target is determined based onphotoluminescence detected from each of the first and second reporters.

37. The method of paragraph 1, wherein the first oligomer includes aphotoluminophore from which the photoluminescence is detected and alsoincludes an energy transfer partner (and/or other modifier of thephotoluminescence) of the photoluminophore.

38. The method of paragraph 37, wherein the energy transfer partner is aquencher.

39. The method of paragraph 37 or 38, wherein the photoluminescence ofthe reporter increases when the first oligomer and the second oligomerbase pair with each other.

40. The method of any of paragraphs 37 to 39, wherein the step ofamplifying a target uses the second oligomer as a primer.

41. The method of any of paragraphs 37 to 40, wherein the secondoligomer does not include an energy transfer partner of thephotoluminophore.

42. The method of any of paragraphs 37 to 41, wherein the step ofamplifying a target does not degrade, or optionally at leaststoichiometrically degrade, the first oligomer.

43. The method of any of paragraphs 37 to 42, wherein the step ofamplifying a target increases the number of first oligomers that are notbound to second oligomers.

44. The method of any of paragraphs 37 to 43, wherein the first oligomerhas a shorter chain of base-containing units than the second oligomer.

45. The method of any of paragraphs 37 to 44, wherein the first oligomerincludes at least one of a second photoluminophore and a second energytransfer partner of the photoluminophore.

46. The method of any of paragraphs 37 to 45, wherein the step ofdetecting the photoluminescence of the reporter is performed while theat least one volume is at a temperature below the melting temperature ofthe reporter.

47. The method of any of paragraphs 37 to 46, wherein the property is alevel of the target.

48. The method of any of paragraphs 37 to 47, wherein the step offorming at least one volume includes a step of forming a plurality ofpartitions that contain the target at partial occupancy.

49. The method of paragraph 48, wherein the partitions are droplets.

50. The method of paragraph 48 or 49, wherein the step of amplifyingincludes a step of exposing the plurality of partitions to a pluralityof thermal cycles, and wherein the partitions are maintainedcontinuously above the melting temperature of the reporter throughoutthe plurality of thermal cycles.

51. The method of any of paragraphs 37 to 50, the reporter being a firstreporter, further comprising a second reporter including a pair ofoligomers capable of base-pairing with one another below a meltingtemperature of the second reporter to affect a photoluminescencedetectable from the second reporter and optionally distinguishable fromthe photoluminescence detectable from the first reporter, wherein thestep of amplifying a target includes a step of extending a forwardprimer provided at least in part by one of the reporters and a step ofextending a reverse primer provided at least in part by the otherreporter, and wherein the property of the target is determined based onphotoluminescence detected from each of the first and second reporters.

52. The method of any of paragraphs 37 to 51, further comprising one ormore limitations of paragraphs 2 to 36 not inconsistent with thelimitations of paragraphs 37 to 51, including but not limited tolimitations describing the placement of photoluminophore(s) and energytransfer partner(s) of the photoluminophore(s), the details ofamplification and detection, the relative alignment of the firstoligomer and second oligomer, the presence of mismatches among the firstand second oligomers and the target, and so on.

53. A method of analysis, the method comprising: (A) forming partitionseach containing a reporter including a first oligomer having aphotoluminophore and also including a second oligomer capable ofbase-pairing with the first oligomer below a melting temperature of thereporter to decrease, by energy transfer, a photoluminescence detectablefrom the photoluminophore; (B) exposing the partitions to a plurality ofthermal cycles, to amplify a target sequence at least in part byextending one or more primers at a temperature above the meltingtemperature of the reporter, wherein the target sequence is present inonly a subset of the partitions; (C) detecting the photoluminescence ofthe photoluminophore for each partition of a plurality of the partitionswhile the partition is at a temperature below the melting temperature ofthe reporter; and (D) determining a property of the target based on thephotoluminescence detected.

54. The method of paragraph 53, wherein the partitions are maintainedcontinuously above the melting temperature of the reporter throughoutthe plurality of thermal cycles.

55. The method of paragraph 53 or paragraph 54, wherein the firstoligomer is a primer included in the one or more primers.

56. The method of any of paragraphs 53 to 55, wherein thephotoluminophore is included in the first oligomer.

57. The method of any of paragraphs 53 to 56, wherein a quencher for thephotoluminophore is included in the second oligomer.

58. The method of any of paragraphs 53 to 57, wherein a quencher for thephotoluminophore is included in the first oligomer.

59. A composition, comprising: a plurality of partitions containing atarget at partial occupancy and amplification reagents to amplify thetarget and also containing a reporter to detect target amplification,the reporter including a first oligomer having a photoluminophore and asecond oligomer that is base-paired with the first oligomer to affect aphotoluminescence detectable from the photoluminophore.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated.

We claim:
 1. A method of analysis, the method comprising: forming amixture including a target; forming a plurality of fluid volumes eachcontaining a portion of the mixture, a reporter, and a pair of primers,wherein only a subset of the plurality of fluid volumes contain at leastone copy of the target, the reporter including a first oligomer having aphotoluminophore and a second oligomer having a quencher for thephotoluminophore, the first oligomer and the second oligomer beingcapable of base-pairing with one another below the melting temperatureof the reporter in the plurality of fluid volumes to quenchphotoluminescence of the photoluminophore, wherein the first oligomerand the second oligomer are not covalently linked to one another;amplifying the target in the plurality of fluid volumes to produce anamplicon by extending the pair of primers at a temperature above themelting temperature of the reporter, wherein amplifying causes cleavageof the second oligomer, and wherein the cleavage increases thephotoluminescence detectable from the photoluminophore of the firstoligomer; detecting photoluminescence from the photoluminophorecontained by each fluid volume of the plurality of fluid volumes whilethe fluid volume is at a temperature below the melting temperature ofthe reporter, wherein fluid volumes of the plurality of fluid volumesthat are positive for the target have more photoluminescence from thephotoluminophore than fluid volumes of the plurality of fluid volumesthat are negative for the target; and determining a level of the targetbased on the photoluminescence detected.
 2. The method of claim 1,wherein the mixture includes the reporter.
 3. The method of claim 1,wherein the mixture includes the pair of primers.
 4. The method of claim1, wherein the plurality of fluid volumes are droplets, and wherein thedroplets are enclosed by an immiscible liquid to create an emulsion. 5.The method of claim 1, wherein determining a level includes determininga number of the plurality of fluid volumes that contain the target orthat do not contain the target based on the photoluminescence detected.6. The method of claim 5, wherein the level is a concentration of thetarget.
 7. The method of claim 1, wherein the second oligomer forms ahybrid with the target having a higher melting temperature than themelting temperature of the reporter.
 8. The method of claim 1, whereinamplifying includes exposing the plurality of fluid volumes to aplurality of thermal cycles, and the plurality of fluid volumes aremaintained continuously above the melting temperature of the reporterthroughout each thermal cycle of the plurality of thermal cycles.
 9. Themethod of claim 1, wherein each of the first and second oligomers has achain of nucleotides, and wherein the chain of nucleotides of the secondoligomer is longer than the chain of nucleotides of the first oligomer.